Genetically modified cells and uses thereof

ABSTRACT

The present invention relates generally to a population of stem cells (e.g., iPSCs or HSCs) that comprise nucleic acids encoding a T cell receptor and a chimeric antigen receptor directed to multiple distinct antigenic determinants, for example two distinct tumour antigenic determinants. The present invention is also directed to a population of T cells that co-express a T cell receptor and a chimeric antigen receptor directed to multiple distinct antigenic determinants, such as two distinct tumour antigenic determinants. The cells of the present invention can be derived from chosen donors whose HLA type is compatible with significant sectors of the populations, and are useful in a wide variety of applications, in particular in the context of the therapeutic treatment of neoplastic conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/778,836, filed May 24, 2018, which claims the benefit of priorityfrom International Patent Application No. PCT/AU2016/051141, filed Nov.23, 2016 and Australian Provisional Patent Application No. 2016901328,filed Apr. 11, 2016 and No. 2015904933, filed Nov. 27, 2015, the entirecontents of which are incorporated herein by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as34247Z_SequenceListing.txt of 36 KB, created on May 9, 2022 andsubmitted to the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to a population of stem cells(e.g., iPSCs or HSCs) that comprise nucleic acids encoding a T cellreceptor and a chimeric antigen receptor directed to multiple distinctantigenic determinants, for example two distinct tumour antigenicdeterminants. The present invention is also directed to a population ofT cells that co-express a T cell receptor and a chimeric antigenreceptor directed to multiple distinct antigenic determinants, such astwo distinct tumour antigenic determinants. The cells of the presentinvention can be derived from chosen donors whose HLA type is compatiblewith significant sectors of the populations, and are useful in a widevariety of applications, in particular in the context of the therapeutictreatment of neoplastic conditions.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

Malignant tumours, or cancers, grow in an uncontrolled manner, invadenormal tissues, and often metastasize and grow at sites distant from thetissue of origin. In general, cancers are derived from one or only a fewnormal cells that have undergone a poorly understood process calledmalignant transformation. Cancers can arise from almost any tissue inthe body. Those derived from epithelial cells, called carcinomas, arethe most common kinds of cancers. Sarcomas are malignant tumours ofmesenchymal tissues, arising from cells such as fibroblasts, musclecells, and fat cells. Solid malignant tumours of lymphoid tissues arecalled lymphomas, and marrow and blood-borne malignant tumours oflymphocytes and other hematopoietic cells are called leukaemias.

Cancer is one of the three leading causes of death in industrialisednations. As treatments for infectious diseases and the prevention ofcardiovascular disease continue to improve, and the average lifeexpectancy increases, cancer is likely to become the most common fataldisease in these countries. Therefore, successfully treating cancerrequires that all the malignant cells be removed or destroyed withoutkilling the patient. An ideal way to achieve this would be to induce animmune response against the tumour that would discriminate between thecells of the tumour and their normal cellular counterparts. However,immunological approaches to the treatment of cancer have been attemptedfor over a century with unsustainable results.

Solid tumours cause the greatest number of deaths from cancer. Solidtumours are not usually curable once they have spread or ‘metastasised’throughout the body. The prognosis of metastatic solid tumours hasimproved only marginally in the last 50 years. The best chance for thecure of a solid tumour relies on early detection followed by the use oflocal treatments such as surgery and/or radiotherapy when the solidtumour is localised and has not spread either to the lymph nodes thatdrain the tumour or elsewhere. Nonetheless, even at this early stage,and particularly if the tumour has spread to the draining lymph nodes,microscopic deposits of cancer known as micrometastases may have alreadyspread throughout the body and will subsequently lead to the death ofthe patient. In this sense, cancer is a systemic disease that requiressystemically administered treatments.

There is a long history of “Golden Bullet” attempts with toxin-loadedantibodies to attack cancers, taking advantage of their capacity topotentially target any specific molecular entity such as carbohydrate,lipid or protein, or combinations thereof. Antibodies, once bound to acancer cell, can engage Complement or FcR+NK/K cells and induce celllysis. Unfortunately antibody treatment of cancer has met generally onlymoderate success, primarily because of low affinity binding, poor lyticefficiency and their brief longevity. Collectively, these compromise theability of antibodies to rapidly destroy cancer cells, increasing therisk of mutation and immune evasion. More recently, there have beenreports of antibody-related therapies including those based onantibodies directed with high affinity to cancer molecules and to immunecheckpoint blockade molecules. Although there are some clinicalsuccesses particularly with the latter, such therapies are stillassociated with various limitations.

Accordingly, common methods of treating cancer continue to follow thelong used protocol of surgical excision (if possible) followed byradiotherapy and/or chemotherapy, if necessary. The success rate of thisrather crude form of treatment is extremely variable but generallydecreases significantly as the tumour becomes more advanced andmetastasises. Further, these treatments are associated with severe sideeffects including disfigurement and scarring from surgery (eg.mastectomy or limb amputation), severe nausea and vomiting fromchemotherapy, and most significantly, damage to normal tissues such asthe hair follicles, gut and bone marrow which is induced as a result ofthe relatively non-specific targeting mechanism of the toxic drugs whichform part of most cancer treatments.

Accordingly, there is an urgent and ongoing need to develop improvedsystemic therapies for cancers, in particular metastatic cancers.

Thymic generation of mainstream T cells is fundamentally required fordefence against infection. This pool of “immune surveillance” T cellspatrols the body to remove damaged or abnormal cells including cancers.Since thymus-based T cell production is characterised by randomgeneration of the T cell receptor (TCR) repertoire, thymopoiesis mustalso include very strict selection processes that eliminate orfunctionally silence those developing thymus T cells with the potentialto attack self. This “self tolerance” therefore restricts autoimmunedisease (Fletcher et al (2011). However, by necessity, this very processcompromises the immune surveillance against cancers—given that non-viralinduced cancers are by definition diseases of “self”. This means thatmany T cells arising in the thymus, which could potentially have beenreactive with tumour-associated antigens may be eliminated before entryinto the blood. At the very least they will be numerically deficient andperhaps have a low affinity TCR. Notwithstanding this, T cells areclearly potentially a major weapon against cancer—the challenges arethus to increase their ability to detect cancer, numerically expand themand retain, or better, enhance their powerful cytolytic capacity. Whileantibodies and T cells are the most logical weapons against cancer,their potential rapid and effective cancer destruction has not beenclinically realized. Advances in immunotherapy have evolved throughgenetically engineering T cells to express a novel chimeric membranereceptor consisting of a cancer antigen binding antibody fragment,coupled cytoplasmically to T cell signal transduction molecules. Thelatter are commonly one or all of the TCR chain, CD 28, or CD40-Ligand(Corrigan-Curay et al (2014); Fedorov et al (2014); Perna et al (2014);Curran et al (2015); Curran et al (2012); Dotti et al (2014); Han et al(2013)). Such chimeric antigen receptor (CAR) expressing T cells (CAR-T)not only harness the two most powerful anticancer weapons of the immunesystem, but also overcome their individual inadequacies. CAR-T retainthe potent, focal, cell lytic capacity and avoid the normal reliance onthe instrinsic TCR to detect very rare “cancer peptide(s)” expressed inHLA clefts. The repertoire of T cells specific to such nominal peptidesis very rare. The antibody portion of the CAR endows the T cells withcancer seeking specificity and overcomes the notoriously poor cancerdestructive efficacy of circulating antibodies. Thus cancer binding ismediated by the antibody domain of the CAR, leading to cytoplasmicsignal transduction, triggering the T cell lytic pathways to destroy thecancer.

Although still in its clinical infancy, numerous CAR-T trials areunderway. As promising as it is though, there are several aspects ofCAR-T technology that are problematic and are preventing its clinicalefficacy to be fully realized. The most obvious is the cytokine stormthat occurs during T cell mediated cancer destruction and is tumour loaddependent. Fever is indicative of cancer destruction, but can lead tosevere clinical side effects unless managed carefully (Davila et al(2014); Casucci et al (2015)). Current management is by cytokinemodulation treatments such as anti-IL6. Further, there exists asignificant problem with the numerical deficiency of generated CAR-Tcells to not only attack the initial cancer, but also to be preserved insufficient supply in case of relapse. Currently, attempts to deal withthis problem are based on the excessive use of proliferation inducingcytokines in vitro. Still further, as effective as CAR-T cells are atattacking cancer, even for CD19⁺ cancers the tumour destruction is not100% effective. While up to 90% responsiveness has been reported forB-ALL, in other CD19⁺ cancers the results are much less effective.Accordingly, despite the encouraging observations in relation to theutility of CAR-T, there are still significant issues to be overcomebefore this technology can take its place as reliable, effective and thenew gold standard in relation to cancer treatment.

In work leading up to the present invention it has been determined,inter alia, that the seemingly disparate problems currently existing inrelation to the effective therapeutic application of CAR-T technologyare resolvable where the CAR-T cells can be derived from transfectedstem cells, such as adult stem cells, rather than transfected thymocytesor other transfected somatic cell types. For example, by transfectingstem cells (such as induced pluripotent stem cells (“iPSCs”) derivedfrom adult somatic cells) with a chimeric antigen receptor, the issue ofproviding sufficient present and future supplies of CAR-T cells directedto a particular tumour is resolved due to the ongoing source of somaticT cells derived from these self-renewing transfected stem cells. Stillfurther, these iPSCs, and hence the CAR-T cells derived from them, canbe prior selected from donors expressing a homozygous HLA haplotype, inparticular homozygous for an HLA type which is expressed widely in thepopulation, thereby providing a means of generating a bank of cellswhich exhibit broad donor suitability. Still further, it has beendetermined that the generation of an iPSC from a T cell which exhibits Tcell receptor specificity directed to an antigen of interest means thatthe gene rearrangements for that TCR specific for the cancer antigenwill be embedded in the iPSC. All T cells induced from that iPSC willretain the anti-cancer TCR specificity. This can be followed bytransfection of such iPSC with a CAR, enabling the subsequentdifferentiation of said iPSC to a T cell, such as a CD4+ or CD8+ T cell,which stably exhibits dual specificity for the antigen to which the CARis directed and a TCR directed to the antigen to which the original Tcell was directed to. Without limiting the present invention to any onetheory or mode of action, it is thought that this is due to the actionsof epigenetic memory. Still further, it has also been determined thatdual specific NKT cells can be similarly generated. Accordingly, therecan be provided an ongoing source of T and NKT cells which areselectively and stably directed to multiple distinct antigenicdeterminants, such as multiple distinct tumour antigenic determinants,thereby enabling a more therapeutically effective treatment step to beeffected.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

As used herein, the term “derived from” shall be taken to indicate thata particular integer or group of integers has originated from thespecies specified, but has not necessarily been obtained directly fromthe specified source. Further, as used herein the singular forms of “a”,“and” and “the” include plural referents unless the context clearlydictates otherwise.

The subject specification contains amino acid sequence informationprepared using the program PatentIn Version 3.5, presented herein afterthe bibliography. Each amino acid sequence is identified in the sequencelisting by the numeric indicator <210> followed by the sequenceidentifier (eg. <210>1, <210>2, etc). The length, type of sequence(protein, etc) and source organism for each amino acid sequence areindicated by information provided in the numeric indicator fields <211>,<212> and <213>, respectively. Amino acid sequences referred to in thespecification are identified by the indicator SEQ ID NO: followed by thesequence identifier (e.g., SEQ ID NO: 1, SEQ ID NO: 2, etc.). Thesequence identifier referred to in the specification correlates to theinformation provided in numeric indicator field <400> in the sequencelisting, which is followed by the sequence identifier (e.g., <400>1,<400>2, etc.). That is SEQ ID NO: 1 as detailed in the specificationcorrelates to the sequence indicated as <400>1 in the sequence listing.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

One aspect of the present invention is directed to a geneticallymodified mammalian stem cell, or a T cell differentiated therefrom,which cell is capable of differentiating to a T cell expressing a TCRdirected to a first antigenic determinant, and comprises a nucleic acidmolecule encoding a chimeric antigen receptor, wherein said receptorcomprises an antigen recognition moiety directed to a second antigenicdeterminant, which antigen recognition moiety is operably linked to a Tcell activation moiety. In some embodiments, the genetically modifiedmammalian stem cell expresses at least one homozygous HLA haplotype.

In another aspect there is provided a genetically modified mammalianstem cell, or a T cell differentiated therefrom, which cell is capableof differentiating to a CD4⁺ T cell expressing a TCR directed to a firstantigenic determinant, and comprises a nucleic acid molecule encoding achimeric antigen receptor, wherein said receptor comprises an antigenrecognition moiety directed to a second antigenic determinant, whichantigen recognition moiety is operably linked to a T cell activationmoiety. In some embodiments, the genetically modified mammalian stemcell expresses at least one homozygous HLA haplotype.

In still another aspect there is provided a genetically modifiedmammalian stem cell, or a T cell differentiated therefrom, which cell iscapable of differentiating to a CD8⁺ T cell expressing a TCR directed toa first antigenic determinant, and comprises a nucleic acid moleculeencoding a chimeric antigen receptor, wherein said receptor comprises anantigen recognition moiety directed to a second antigenic determinant,which antigen recognition moiety is operably linked to a T cellactivation moiety. In some embodiments, the genetically modifiedmammalian stem cell expresses at least one homozygous HLA haplotype.

In a further aspect there is provided a genetically modified mammalianstem cell, or a T cell differentiated therefrom, which cell is an iPSC(induced pluripotent stem cell) or an HSC (haemopoietic stem cell), iscapable of differentiating to a T cell expressing a TCR directed to afirst antigenic determinant, and comprises a nucleic acid moleculeencoding a chimeric antigen receptor, wherein said receptor comprises anantigen recognition moiety directed to a second antigenic determinant,which antigen recognition moiety is operably linked to a T cellactivation moiety. In some embodiments, the genetically modified stemcell such as iPSC or HSC expresses at least one homozygous HLAhaplotype.

In accordance with this aspect of the invention, in one embodiment, thestem cell (e.g., iPSC) is derived from a cell in which the TCR geneshave undergone re-arrangement.

In another embodiment, said stem cell (e.g., iPSC) is derived from a Tcell or thymocyte expressing an αβ TCR.

In still another embodiment, said stem cell (e.g., iPSC) is derived froma T cell or thymocyte expressing a γδ TCR.

In yet another embodiment, said stem cell (e.g., iPSC) is derived from aT cell or thymocyte expressing a TCR directed to said first antigenicdeterminant, i.e., the same antigenic determinant to which the TCRexpressed on a T cell derived from said stem cell (e.g., iPSC) isdirected.

In still another embodiment, said stem cell (e.g., iPSC) is derived froma T cell or thymocyte that is CD8⁺.

In yet another embodiment, said stem cell (e.g., iPSC) is derived from aT cell or thymocyte that is CD4⁺.

In one embodiment, the stem cell (e.g., iPSC or HSC) is capable ofdifferentiating into a CD4⁺ T cell expressing a TCR directed to a firstantigenic determinant. In another embodiment, the stem cell (e.g., iPSCor HSC) is capable of differentiating into a CD8⁺ T cell expressing aTCR directed to a first antigenic determinant.

In still another further aspect there is provided a genetically modifiedmammalian stem cell, or a T cell differentiated therefrom, which cell iscapable of differentiating to a T cell expressing a TCR directed to afirst antigenic determinant, and comprises a nucleic acid moleculeencoding a chimeric antigen receptor, wherein said receptor comprises anantigen recognition moiety directed to a second antigenic determinant,which antigen recognition moiety is operably linked to a T cellactivation moiety, and wherein said antigenic determinants are selectedfrom tumour antigens, microorganism antigens or autoreactive immune cellantigens. In some embodiments, the genetically modified mammalian stemcell expresses at least one homozygous HLA haplotype.

In one embodiment, said stem cell is an iPSC. In another embodiment, thestem cell is an HSC.

In another embodiment, said stem cell is capable of differentiating to aCD4⁺ T cell or a CD8⁺ T cell.

In still another embodiment, said TCR is an αβ TCR.

In yet still another embodiment, said stem cell (e.g., iPSC) is derivedfrom a T cell or thymocyte, preferably a CD8⁺ T cell or thymocyte. Insome embodiments, said stem cell (e.g., iPSC) is derived from a CD8⁺ Tcell or thymocyte expressing a TCR directed to said first antigenicdeterminant, i.e., the same antigenic determinant to which the TCRexpressed on a T cell derived from said stem cell (e.g., iPSC) isdirected.

In yet another aspect there is provided a genetically modified mammalianstem cell, or a T cell differentiated therefrom, which cell is capableof differentiating to a T cell expressing a TCR directed to a firsttumour antigenic determinant, and comprises a nucleic acid moleculeencoding a chimeric antigen receptor, wherein said receptor comprises anantigen recognition moiety directed to a second tumour antigenicdeterminant, which antigen recognition moiety is operably linked to a Tcell activation moiety, and wherein said first antigenic determinant isselected from TCR recognized peptides such as WT-1 or EbvLMP2, and saidsecond antigenic determinant is selected from, for example, TAG-72,CD19, MAGE, or CD47. In some embodiments, the genetically modifiedmammalian stem cell expresses at least one homozygous HLA haplotype.

The genetically modified mammalian stem cells (e.g., iPSCs or HSCs)disclosed herein are capable of differentiating to a T cell expressing aTCR directed to a first antigenic determinant (e.g., a first tumourantigenic determinant), and comprises a nucleic acid molecule encoding achimeric antigen receptor which comprises an antigen recognition moietydirected to a second antigenic determinant (e.g., a second tumourantigenic determinant), operably linked to a T cell activation moiety.That is, the genetically modified stem cells (e.g., iPSCs or HSCs)disclosed herein are capable of differentiating into T cells directed tomultiple, i.e., at least two (namely two or more) antigenicdeterminants. In some embodiments, the genetically modified mammalianstem cell expresses at least one homozygous HLA haplotype.

Accordingly, in a further aspect, there is provided a geneticallymodified mammalian stem cell capable of differentiating into a T celldirected to more than two antigenic determinants.

In accordance with this aspect of the invention, in some embodiments,the genetically modified mammalian stem cell (e.g., iPSC or HSCs) iscapable of differentiating to a T cell expressing a TCR directed to afirst antigenic determinant, and comprises multiple (i.e., two or more)nucleic acid molecules encoding multiple chimeric antigen receptors,wherein each chimeric antigen receptor comprises an antigen recognitionmoiety directed to an antigenic determinant, which antigen recognitionmoiety is operably linked to a T cell activation moiety. In someembodiments, the genetically modified mammalian stem cell expresses atleast one homozygous HLA haplotype.

In one embodiment, the multiple antigenic determinants which themultiple chimeric antigen receptors are directed to are each distinctfrom said first antigenic determinant to which the TCR expressed on a Tcell derived from said stem cell is directed. In another embodiment, themultiple antigenic determinants which the multiple chimeric antigenreceptors are directed to are distinct, one from another, and are alsodistinct said first antigenic determinant to which the TCR expressed ona T cell derived from said stem is directed.

In one embodiment, the multiple CAR-encoding nucleic acids are includedin one contiguous nucleic acid fragment. For example, the multipleCAR-encoding nucleic acids are placed in one construct or vector whichis transfected into a cell to generate a genetically modified mammalianstem cell comprising the multiple CAR-encoding nucleic acids. In aspecific embodiment, the multiple CAR encoding nucleic acids can belinked to each other within one expression unit and reading frame (forexample, by utilizing a self-cleaving peptide such as P2A), such thatone single polypeptide comprising multiple CAR polypeptide sequences isinitially produced and subsequently processed to produce multiple CARs.In another embodiment, the multiple CAR-encoding nucleic acids areplaced in separate vectors which are used in transfection to generate agenetically modified mammalian stem cell comprising the multipleCAR-encoding nucleic acids. Examples of CAR-encoding nucleic acidconstructs are depicted in FIG. 11, and exemplary sequences for a CARand various domains suitable for use in a CAR are provided in SEQ IDNOS: 1-2 and 7-20.

Further in accordance with the aspect of the invention providing agenetically modified mammalian stem cell capable of differentiating intoa T cell directed to more than two antigenic determinants, in otherembodiments, the genetically modified mammalian stem cell (e.g., iPSC orHSC) (which optionally expresses at least one homozygous HLA haplotype),is capable of differentiating to a T cell expressing a TCR directed to afirst antigenic determinant, comprises a nucleic acid molecule encodinga chimeric antigen receptor which comprises an antigen recognitionmoiety directed to a second antigenic determinant, operably linked to aT cell activation moiety, and further comprises a nucleic acid moleculeencoding an antigen-binding receptor which comprises an antigenrecognition moiety directed to a third antigenic determinant. Accordingto these embodiments, such genetically modified stem cell is capable ofdifferentiating into a T cell directed to multiple antigenicdeterminants, preferably multiple antigenic determinants that aredistinct one from another. Additional antigenic specificity can beprovided by employing multiple CAR-encoding nucleic acids as describedherein, and/or utilizing multiple nucleic acids encoding antigen bindingreceptors.

In one embodiment, the antigen-binding receptor is a non-signallingantigen-binding receptor; namely, the receptor is anchored to the cellsurface and binds to the third antigenic determinant, but does nottransduce signal into the cytoplasmic part of the cell. In oneembodiment, the antigen-binding receptor comprises an antigenrecognition moiety directed to a third antigenic determinant, operablylinked to a transmembrane domain, but lacks a T cell activation moiety.

In a specific embodiment, the antigen-binding receptor is anon-signalling antigen-binding receptor directed to CD47. For example,the antigen-binding receptor is a non-signalling CD47-binding molecule,e.g., a truncated, CD47-binding molecule.

Accordingly, there is provided a genetically modified mammalian stemcell (e.g., iPSC or HSC), or a T cell differentiated therefrom, whichcell is capable of differentiating to a T cell expressing a TCR directedto a first antigenic determinant, comprises (i) a nucleic acid moleculeencoding a chimeric antigen receptor, wherein said receptor comprises anantigen recognition moiety directed to a second antigenic determinant,which antigen recognition moiety is operably linked to a T cellactivation moiety and (ii) a nucleic acid molecule encoding anon-signalling CD47-binding molecule, e.g., a truncated, CD47-bindingmolecule. In some embodiments, the genetically modified mammalian stemcell (e.g., iPSC or HSC) expresses at least one homozygous HLAhaplotype.

In another aspect there is provided a method of making a geneticallymodified mammalian stem cell (such as an iPSC or HSC) disclosed herein.

In one embodiment, the subject method comprises obtaining a mammalianstem cell (such as an iPSC or HSC) that is capable of differentiating toa T cell expressing a TCR directed to a first antigenic determinant,which stem cell (e.g., iPSC or HSC), in one embodiment, expresses atleast one homozygous HLA haplotype; and introducing into the stem cell(e.g., via transfection) one or more nucleic acid molecules encoding oneor more chimeric antigen receptors, each chimeric antigen receptorcomprising an antigen recognition moiety directed to an antigenicdeterminant, which antigen recognition moiety is operably linked to a Tcell activation moiety. In another embodiment, the method furthercomprises introducing into the stem cell (e.g., via transfection) one ormore nucleic acid molecules encoding one or more antigen-bindingreceptors (e.g., non-signalling antigen-binding receptors), eachantigen-binding receptor comprising an antigen recognition moietydirected to an antigenic determinant. As further disclosed herein, themultiple receptor-encoding nucleic acids can be introduced by way of asingle vector or separate vectors.

In another embodiment, the subject method comprises obtaining a T cellor thymocyte (preferably CD8+ T cell or thymocyte) which expresses a TCRdirected to a first antigenic determinant, and which, in one embodiment,also expresses at least one homozygous HLA haplotype; introducing intothe T cell or thymocyte one or more nucleic acid molecules encoding oneor more chimeric antigen receptors, each chimeric antigen receptorcomprising an antigen recognition moiety directed to an antigenicdeterminant, which antigen recognition moiety is operably linked to a Tcell activation moiety; and deriving a stem cell (e.g., iPSC) from the Tcell or thymocyte. In another embodiment, the method further comprises,before the step of deriving a stem cell from the T cell or thymocyte,introducing into the T cell or thymocyte one or more nucleic acidmolecules encoding one or more antigen-binding receptors (e.g.,non-signalling antigen-binding receptors), each antigen-binding receptorcomprising an antigen recognition moiety directed to an antigenicdeterminant.

In still another embodiment, the subject method comprises obtaining anHSC (e.g., from the bone marrow or blood) which, in some embodiments,expresses at least one homozygous HLA haplotype; introducing to the HSC(i) one or more nucleic acids encoding a TCR directed to a firstantigenic determinant, (ii) one or more nucleic acid molecules encodingone or more chimeric antigen receptors, each chimeric antigen receptorcomprising an antigen recognition moiety directed to an antigenicdeterminant that is different from said first antigenic determinant,which antigen recognition moiety is operably linked to a T cellactivation moiety; and optionally (iii) one or more nucleic acidmolecules encoding one or more antigen-binding receptors (e.g.,non-signalling antigen-binding receptors), each antigen-binding receptorcomprising an antigen recognition moiety directed to an antigenicdeterminant that is different from said first antigenic determinant anddifferent from the antigen determinant(s) to which the chimeric antigenreceptor(s) is(are) directed. As disclosed herein, the multiplereceptor-encoding nucleic acids can be introduced by way of a singlevector or separate vectors. Such genetically modified HSC can be used togenerate T cells having specificity to multiple antigenic determinants.

In a further aspect there is provided a T cell that expresses a TCRdirected to a first antigenic determinant, and expresses one or morechimeric antigen receptors, wherein each receptor comprises an antigenrecognition moiety directed to an antigenic determinant, which antigenrecognition moiety is operably linked to a T cell activation moiety. Insome embodiments, the T cell further expresses an antigen-bindingreceptor which comprises an antigen recognition moiety directed anantigenic determinant. In some embodiments, the T cell provided thereinexpresses at least one homozygous HLA haplotype.

In another aspect there is provide a method for making a T cell thatexpresses a TCR directed to a first antigenic determinant, and expressesone or more CARs wherein each CAR comprises an antigen recognitionmoiety directed to an antigenic determinant, which antigen recognitionmoiety is operably linked to a T cell activation moiety, and optionallyalso expresses one or more non-signalling antigen-binding receptors eachof which comprises an antigen recognition moiety directed to anantigenic determinant. In some embodiments, the method provided hereinis directed to making a T cell that expresses at least one homozygousHLA haplotype.

Another aspect of the present invention is directed to a method oftreating a condition characterised by the presence of an unwantedpopulation of cells in a mammal, said method comprising administering tosaid mammal an effective number of stem cells or T cells, ashereinbefore described.

In one embodiment, said condition is a neoplastic condition, amicroorganism infection (such as HIV, STD or antibiotic resistantbacteria), or an autoimmune condition.

According to this embodiment, there is provided a method of treating aneoplastic condition, said method comprising administering to saidmammal an effective number of stem cells, or T cells, as hereinbeforedefined wherein said TCR is directed to a first tumour antigenicdeterminant and said CAR is directed to a second tumour antigenicdeterminant.

In still another embodiment, said first tumour antigenic determinant isWT-1.

In another embodiment, said second tumour antigenic determinant isTAG-72, CD19, MAGE, or CD47.

Yet another aspect of the present invention is directed to the use ofstem cells or T cells, as hereinbefore defined in the manufacture of amedicament for the treatment of a condition characterised by thepresence of an unwanted population of cells in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIGS. 1A-10. Stimulation and expansion of cytotoxic T cells expressing aTCR specific for Wilm's Tumor 1 (WT-1) antigen. Cells were isolated fromwhole blood peripheral blood mononuclear cells (PBMCs). Cells were gatedon single cells (A, F, K) where the scatter plot is also depicted (B, G,L), followed by CD3 positive cells (conjugated to APCCy7; C, H, M),followed by CD8 (conjugated to PECy7) and CD4 (conjugated to PerCp; D,I, N), and finally on CD8 cells alone (E, J, O). WT-1 staining wascarried out using HLA-A02 tetramer specific for the WT-1₃₇ peptide.Representations are from two separate patients (patient 1 A-E; patient 2F-J) that are HLA-A02 positive and compared to a fluorescence minus one(FMO; this stain lacks the WT-1 tetramer stain, showing the specificitystain of WT-1₃₇, K-O). Proportions shown are a percentage of CD3+ cells.The percentage of WT-1 TCR T cells increased to 1.5% and 4.5% for thetwo samples; in unstimulated PBMC these cells are very low (below thelevel of detection using the tetramer technology herein). In otherstudies (e.g., Schmeid et al (2015)) they are as few 1 per 10⁻⁶ of CD8+cells (range 3×10⁻⁷ to 3×10⁻⁶ cells).

FIGS. 2A-2G. CD8+ Cytotoxic T cells with TCR specific for Wilm's Tumor 1(WT-1) antigen are functional. Function is represented by production ofinterferon gamma (IFN-γ) (Ghanekar et al 2001). IFN-γ expression wasfound after WT-1 specific stimulation. Activated cells were gated on theCD8+ and HLA-A02 tetramer to the WT-1₃₇ peptide—PE conjugatedfluorochrome. These cytotoxic T cells with TCR specific for WT-1, whenstimulated with WT-1, demonstrated intracellular cytokine stain of IFN-γ(conjugated to the pacific blue fluorochrome). Representations are fromtwo separate patients (Wt-1 #1 and WT-1 #2) (patient 1: A-B; patient 2:C-D) that are HLA-A02 positive and compared to a fluorescence minus one(FMO; E-F; this stain lacked the WT-1 tetramer stain, showing thespecificity stain of WT-1₃₇ (G). Proportions shown are a percentage ofWT1+CD8+ cells. Over 80% of the WT-1 TCR T cells produced IFNγ.

FIG. 2H. Addition of the LAG 3 inhibitor (IMP 321) increases thefrequency of WT-1 specific T cells after 4 days stimulation. In thisexperiment, purified but unseparated cord blood mononuclear cells wereplated either alone, with either anti CD28 alone, with WT-1 peptide(Miltenyi BioTech) and CD28 μg/ml) or with WT-1 peptide plus IMP 321,for 24 hours and 4 days. No effects were observed by 24 hours (data notshown) but, consistent with the kinetics of IMP 321 effect on activationdendritic cells (Brigone et al (2007)), there was a doubling of WT-1specific CD8+ T cells after 4 days.

FIG. 3. Production of iPSC from cancer specific (eg WT-1) TCR T cells.Cancer antigen specific T cells are extremely rare in normal blood; theyare revealed by stimulation in vitro with WT-1 peptide bound toautologous B cells acting as antigen presenting cells (formed intolymphoblast cells lines (LCL) using EBV), in the presence of cytokines.Cancer antigen specific T cells are shown as double labelled with CD8(for cytotoxic T cells) and tetramer for HLA-WT-1 binding to the TCR ofthese CD8+ cells. These cells were then converted into iPSC using theYamanaka reprogramming factors. The rearranged TCR genes specific forWT-1 were embedded in the TCR locus of the iPSCs.

FIG. 4. Morphological progression of iPSC colonies to haemopoieticlineage and lymphoid progenitors after culture for 1, 5, 9 and 13 dayson OP9 support cells. Note large numbers of single haemopoietic-likecells by day 13.

FIG. 5. Flow cytometric analysis of iPSC-derived cells after culture for13 days on OP9 cells, clearly shows evidence of haemopieticspecialisation with the presence of haemopoietic stem cells (HSC)(CD34+CD43+).

FIG. 6. Flow cytometry for HSC in iPSC-derived cells after 13 daysculture on OP9 cells followed by 9 days culture on OP9 DL-L1 cells.Cells were gated on viability, CD45 expressions, single cells thenexamined for HSC content by staining for CD34 and CD43. Note thereduction in HSC from >90% pre OPDL-L1 culture (FIG. 5), to ˜60% after 9days culture on OP9DL-L1 cells.

FIG. 7. Flow cytometry for T cell development of iPSC-derived cellsafter 13 days culture on OP9 cells followed by 9 days culture on OP9DL-L1 cells. There is clear evidence of commitment to the T cell lineagewith expression of CD5 and CD7 and the first stages of thymocytedevelopment with immature (i.e., lacking CD3; data not shown) CD4+,CD8+“single positive” cells and CD4+CD8+“double positive” cells.

FIG. 8. Flow cytometry for HSC and T cell differentiation iniPSC-derived cells after 13 days culture on OP9 cells followed by 16days culture on OP9 DL-L1 cells. Immature T cells expressing CD4 and/orCD8 were still clearly present and there was further reduction of HSCfrom ˜60% to ˜25%. Most importantly mature CD8+ cells were present andexpressed CD3, αβ TCR and the CD8β chain (in addition to CD8a—not shown)

FIG. 9. Schematic representation of the induction of WT-1 specific TCR,CD8αβ T cells from iPSC derived from in vitro expanded WT-1 specific TCRT cells. The treatment of the CD4+CD8+ cells with (low levels) anti CD3antibody mimic the signalling that occurs within the thymus duringpositive selection; this increased CD8+ T cells expressing both the CD8αand CD8β chains.

FIG. 10. WT-1 specific TCR, CD8αβ T cells induced from iPSC derived fromin vitro expanded WT-1 specific TCR T cells, retained full function(e.g., cytotoxicity to WT-1 expressing targets) equivalent to theoriginal T cells. The effector:target ratio was 3:1; gradedconcentrations of WT-1 peptides were tested.

FIG. 11. Schematic diagram of chimeric antigen receptor andantigen-binding receptor constructs. A panel of Chimeric AntigenReceptor (CAR) constructs have been developed—with scFv for either TAG72 or CD19 (as a positive control). The constructs used either human CD8or CD28 as hinge and transmembrane regions and CD28, CD3ζ chain or 4-1BBcytoplasmic activation signalling domains. P2A is a signal sequencedirecting proteolytic cleavage, which in the top five constructs shownin FIG. 11 releases EGFP as a fluorescent reporter of expression, and inthe lower (sixth) construct shown in FIG. 11, releases a second CARreceptor construct shown as Leader (CD8)-scFv (anti-CD47)-hinge/TM(CD28)-endodomain tail (CD8) in which the Leader will be processed torelease the anti-CD47 scFv on the surface anchored by the hinge/TM andthe endodomain tail contains no signalling sequences. Any CD47-bindingectodomain could be used for the purpose of binding to CD47 on targetcells, including for example SIRP-alpha. The hinge region may containcysteine residues to direct dimerization by disulphide bond formationbetween adjacent hinge domains, which is characteristic of the naturalCD8 hinge, or may have the cysteine residues substituted by otherresidues, such as serine, which do not form disulphide bonds and do notform covalently stabilised dimers. Exemplary sequences of CAR andCD47-binding receptor, as well as various domain sequences suitable foruse in constructing a CAR or an antigen-binding receptor, are set forthin SEQ ID NOS: 1-20.

FIG. 12. Retrovirus Transformation scheme. Schematic of the processesundertaken for generating CAR containing retroviral constructs. The CARconstruct is cloned into the pSAMEN plasmid vector and is linked to thefluorescent reporter EGFP by a P2A self-cleaving polypeptide to separatethe CAR and reporter. When transduction of the cell is successful, theP2A is expressed and cleaved, and the EGFP is identified by flowcytometry and immunofluorescence microscopy.

FIG. 13. Lentivirus Transformation scheme. Schematic of the processesundertaken for generating CAR containing lentiviral constructs. The CARconstruct is cloned into the pWP1 plasmid vector and is linked to thefluorescent reporter EGFP by a P2A self-cleaving polypeptide to separatethe CAR and reporter. When transduction of the cell is successful theP2A is expressed and cleaved, and the EGFP identified by flow cytometryand immunofluorescence microscopy.

FIG. 14A. Schematic of normal second generation CAR structure. scFvbinding domains to target antigens; hinge region (stalk) allowingintegration of the CAR into the plasma membrane (length of hinge candifferentially influence scFv binding to target cells); cytoplasmicsignalling domains which induce T cell activation upon engagement of thescFv. The CAR structure is shown as a dimer, stabilised by disulphidebonds between adjacent cysteine residues in the hinges region.

FIG. 14B. Schematic of a non-signalling antigen-binding receptor, atruncated CD47 “attachment stalk”. Structure shows scFv domains orsingle V-domains for CD47 antigen binding, attached to a hinge andtransmembrane region but no signalling domains are present in theendodomain. This construct would allow increased binding affinity of theCAR-T cell to the cancer cells expressing high levels of CD47. Whilethis receptor could also bind to normal cells which express lower levelsof CD47, there would be no signal transduction and hence no damage tothe normal cells. The Hinge region may contain cysteine residues todirect dimerization by disulphide bond formation between adjacent hingedomains, or may have the cysteine residues substituted by otherresidues, such as serine, which do not form disulphide bonds and do notform covalently stabilised dimers.

FIG. 15. Flow cytometry analysis of CAR transduced human PBMC derivedCD3+ T cells demonstrating successful transduction with the TAG72Lentivirus CAR construct (20.8% positive compared to <0.1% in thecontrols) and CD19 lentivirus CAR construct (33.9% positive).

FIG. 16. Western blot analysis confirming protein expression in TAG 27and CD19 CAR-transfected T cells.

FIG. 17. TAG-72 CAR-T mediated killing of ovarian cancer (TAG72+) targetcells. Effector:target ratio (E:T)=1:1. TAG-72 CAR-T effector cells (GFPpositive cells) developed from CD3 activated normal blood T cells, wereisolated as >95% pure via FACS and subsequently stimulated for enhancecytolytic activity for 72 h in the presence of immobilised αCD³/αCD28and IL-2 before use. Change in cell impedance (represented here as thearbitrary unit Cell Index) was monitored over 40 h and compared tostimulated non-transduced CD3^(+ve) cells isolated from PBMCs andstimulated vector only CAR-T cells. TAG72 CAR-T cells showed the highestkilling however CD3/CD28 activated non-CAR-T cells also showed killing,albeit to a much lesser degree.

FIG. 18. Determining the specificity of TAG-72 CAR-T killing. TAG-72 andCD19 CAR-T respectively were isolated via FACS and immediately added toTAG-72^(hi)/CD19^(low) target cells without in vitro stimulation(E:T=5:1). Change in cell impedance (represented here as the arbitraryunit Cell Index) was monitored over 15 h. TAG-72 CAR-T cells showedstrong killing of the cell line. CD19 CAR-T cells were the same asnon-CAR T cell controls.

FIGS. 19A-19B. Flow cytometry analysis of CAR transduction of WT-1specific TCR CD8+ T cells derived from iPSC produced from WT-1 specificT cells. FIG. 19A. WT-1 specific TCR T cells were successfullytransduced with the TAG72 Lentivirus CAR construct (31.3% positivecompared to <0.1% in the controls). FIG. 19B. WT-1 specific TCR T cellsderived from iPSC formed from WT-1 specific TCR T cells successfullytransduced with dual specificity CAR construct for TAG 72 plusnon-signalling truncated CD47 (55% transduced); transduced with TAG 72alone 32%. These transduced T cells contained 3 anti-cancerspecificities: WT-1 (TCR); TAG72 (CAR); truncated non-signalling CD47.

FIG. 20A-20I. Cytotoxic function of WT-1 specific TCR T cells, and dualspecific TAG 72 CAR/WT-1 TCR T cells. WT-1 specific TCR T cells and dualspecific TAG72 CAR/WT-1 TCR T cells were incubated in monolayer cultureswith the ovarian cancer cell line CAOV4 to for 24 hours to assesscytotoxicity. Despite the low effector:target ratio of 2:1 (necessarybecause of the low numbers of effectors obtained), there was specifickilling with WT-1 TCR T cells and this was increased further withtransduction with the TAG72 CAR. The technique is based on AquaAminewhich stains amines within the cell. When a cell dies or is dying thecompromised cell membrane allows the dye to infiltrate the cell andstain the amines more intensely. Cell cytotoxicity is therefore depictedby an increased staining intensity of cellular amines. Note: live cellswill still give some (albeit low) positive staining because some aminesreside on the cell surface. A, D, G: CAOV4 cancer cells alone. B, E, H:CAOV4 cancer cells incubate with WT-1 TCR T cells. C, F, I: Dualspecific TAG 72 CAR/WT-1 TCR T cells incubated with CAOV4 ovarian cancercells. D, E, F: Aqua amine levels on gated CD3-ve cells (i.e., CAOVA4).Phase contrast images of G: Cancer cells alone, H: non-CAR transfectedWT-1 TCR cells with cancer cells, and I: TAG-72 transfected WT-1 TCR Tcells and cancer cells. 40× magnification. WT-1 TCR T cells causedapproximately 10% killing (above background); TAG72 CAR-T cells causedan additional 10% killing (i.e., approximately 20% above background).Dual anti-cancer killing mechanisms are additive.

FIGS. 21A-21B. CAR transduction of iPS. Day 5 of growth on MEF feederlayers, 4 days after incubation with CAR lentivirus. CAR+ transduction(green) of TAG72, CD19 and GFP virus were overlayed on bright fieldimages at 20× magnification. Non-transduced controls did not display anyGFP signal. Images of iPSC colonies at 4× magnification demonstrate thepresence of iPSC colonies on MEF feeder layers. In each system it isnoted that some of the iPSC colonies appeared to have begun tospontaneously differentiate. Transduced fibroblast-derived iPSC aredepicted in FIG. 21A. FIG. 21B demonstrates the successful transductionof WT-1 T cell derived iPSCs with TAG72 CAR. These iPSCs were thereforesuccessfully imprinted for both WT-1 TCR and TAG 72 specificity.

FIG. 22. Flow cytometric analysis of Chimeric Antigen Receptortransduction of iPSC. These iPSC are derived from adult fibroblasts butcan be from any origin including non-selected T cells, CD8+ T cells orcancer antigen specific (e.g., WT-1) T cells. There is clearly apopulation of fluorescent iPSC successfully transduced by TAG 72 orCD19. Overlay of the transduced cells compared to non-transducedcontrols is shown in FIG. 23.

FIG. 23. Overlay of dot plots comparing non-transduced control cells(blue) to transduced iPSC cultures (green). Events within the GFP+ gatedemonstrate successful transduction and are presented as percentfrequency of non-debris events.

FIG. 24. Reformation of CAR-Transduced iPSC colonies after FACS sorting.CAR-transduced iPSC can be isolated by flow cytometry (GFP positivefluorescence) and replated to form stable colonies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination thatdual TCR/CAR expressing T cells, directed to two distinct antigenicdeterminants, can be consistently and stably generated by, for example,transfecting a CAR cassette into an iPSC derived from a T cellexhibiting TCR specificity directed to an antigenic determinant ofinterest. By virtue of the actions of epigenetic memory, a T celldifferentiated from this iPSC has been found to stably express both theTCR specificity of the somatic T cell from which the iPSC was derived,and a CAR directed to a distinct antigenic determinant. Specificity toadditional antigenic determinants can be achieved by introducing intocells additional nucleic acid(s) encoding a molecule(s) that bind(s) tosuch additional antigenic determinant(s). Such multi-specificity cellthereby provides a more effective therapeutic outcome than currentlyavailable. These determinations have therefore now enabled thedevelopment of an ongoing source of stably transformed dual antigenspecific T cells, in particular cytotoxic CD8+αβ TCR T cells, for use inthe context of any disease condition which is characterised by anunwanted cellular population, such as a neoplastic condition, a viralinfection, bacterial infection or an autoimmune condition. This finding,and the generation of cells based thereon, have now facilitated theimprovement of therapeutic treatment regimes directed to treating suchconditions, in particular neoplastic conditions such as solid tumours orblood cancers (e.g., leukaemias), including metastatic disease.

Accordingly, one aspect of the present invention is directed to agenetically modified mammalian stem cell, or a T cell differentiatedtherefrom, which cell is capable of differentiating to a T cellexpressing a TCR directed to a first antigenic determinant, andcomprises a nucleic acid molecule encoding a chimeric antigen receptor,wherein said receptor comprises an antigen recognition moiety directedto a second antigenic determinant, which antigen recognition moiety isoperably linked to a T cell activation moiety. In some embodiments, thegenetically modified mammalian stem cell expresses at least onehomozygous HLA haplotype.

Reference to a “T cell” should be understood as a reference to any cellcomprising a T cell receptor. In this regard, the T cell receptor maycomprise any one or more of the α, β, γ or γ chains. As would beunderstood by the person of skill in the art, NKT cells also express a Tcell receptor and therefore dual specific NKT cells can also begenerated according to the present invention. The present invention isnot intended to be limited to any particular sub-class of T cell,although in a preferred embodiment the subject T cell expresses an α/βTCR dimer. Still more preferably, said T cell is a CD4⁺ helper T cell, aCD8⁺ killer T cell, or an NKT cell. Without limiting the presentinvention to any one theory or mode of action, CD8⁺ T cells are alsoknown as cytotoxic cells. As a major part of the adaptive immune system,CD8⁺ T cells scan the intracellular environment in order to target anddestroy, primarily, infected cells. Small peptide fragments, derivedfrom intracellular content, are processed and transported to the cellsurface where they are presented in the context of MHC class Imolecules. However, beyond just responding to viral infections, CD8+ Tcells also provide an additional level of immune surveillance bymonitoring for and removing damaged or abnormal cells, includingcancers. CD8⁺ T cell recognition of an MHC I presented peptide usuallyleads to either the release of cytotoxic granules or lymphokines or theactivation of apoptotic pathways via the FAS/FASL interaction to destroythe subject cell. CD4⁺ T cell, on the other hand, generally recognisepeptide presented by antigen presenting cells in the context of MHCclass II, leading to the release of cytokines designed to regulate the Bcell and/or CD8+ T cell immune responses. Accordingly, unlike cytotoxicT cells, T helper cells do not directly kill unwanted cells, such ascancer cells, although they can augment such a response, to the extentthat it is effected by cytotoxic T cells and/or antibody based clearancemechanisms.

Natural killer T (NKT) cells are a specialised population of T cellsthat express a semi-invariant T cell receptor (TCR αβ) and surfaceantigens typically associated with natural killer cells. The TCR on NKTcells is unique in that it recognizes glycolipid antigens presented bythe MHC I-like molecule CD1d. Most NKT cells express an invariant TCRalpha chain and one of a small number of TCR beta chains. The TCRspresent on type I NKT cells recognise the antigenalpha-galactosylceramide (alpha-GalCer). Within this group,distinguishable subpopulations have been identified, including CD4⁺CD8⁻cells, CD4⁻CD8⁺ cells and CD4″/CD8″ cells. Type II NKT cells (ornoninvariant NKT cells) express a wider range of TCR α chains and do notrecognise the alpha-GalCer antigen. NKT cells produce cytokines withmultiple, often opposing, effects, for example either promotinginflammation or inducing immune suppression including tolerance. As aresult, they can contribute to antibacterial and antiviral immuneresponses, promote tumour-related immunosurveillance, and inhibit orpromote the development of autoimmune diseases. Like natural killercells, NKT cells can also induce perforin-, Fas-, and TNF-relatedcytoxicity. Accordingly, reference to the genetically modified T cellsof the present invention should be understood to include reference toNKT cells.

Since thymus-based T cell production is characterised by randomgeneration of the T cell receptor (TCR) repertoire, thymopoiesis mustalso include very strict selection processes that eliminate orfunctionally silence those developing thymus T cells with the potentialto attack self. This “self tolerance” therefore reduces the potentialfor autoimmune disease. However, by necessity, this very processcompromises the immune surveillance against cancers—given that non-viralinduced cancers are by definition diseases of “self”. This means thatmany T cells arising in the thymus, which could potentially have beenreactive with tumour-associated antigens, may be eliminated before entryinto the blood. At the very least they will be numerically deficient andperhaps express a low affinity TCR.

In one embodiment there is provided a genetically modified mammalianstem cell, or a T cell differentiated therefrom, which cell is capableof differentiating to a CD4⁺ T cell expressing a TCR directed to a firstantigenic determinant, and comprises a nucleic acid molecule encoding achimeric antigen receptor, wherein said receptor comprises an antigenrecognition moiety directed to a second antigenic determinant, whichantigen recognition moiety is operably linked to a T cell activationmoiety. In one embodiment, the genetically modified mammalian stem cellexpresses at least one homozygous HLA haplotype.

In another embodiment there is provided a genetically modified mammalianstem cell, or a T cell differentiated therefrom, which cell is capableof differentiating to a CD8⁺ T cell expressing a TCR directed to a firstantigenic determinant, and comprises a nucleic acid molecule encoding achimeric antigen receptor, wherein said receptor comprises an antigenrecognition moiety directed to a second antigenic determinant, whichantigen recognition moiety is operably linked to a T cell activationmoiety. In one embodiment, the genetically modified mammalian stem cellexpresses at least one homozygous HLA haplotype.

In some embodiments, the genetically modified cell of the presentinvention, e.g., the genetically modified stem cell (such as an iPSC oran HSC) or T cell, is homozygous for at least one HLA haplotype. Withoutlimiting the present invention to any one theory or mode of action, themajor histocompatibility complex (MHC) represents a set of cell surfacemolecules, the major function of which is to bind peptide fragmentsderived from antigens and to present them to T cells. The MEW genefamily is divided into three subgroups: class I, class II and class III.Class I MHC molecules express (32 subunits and therefore can only berecognised by CD8 co-receptors. Class II MHC molecules express no (32subunits and can therefore be recognised by CD4 co-receptors. In thisway, MHC molecules regulate which type of lymphocytes may bind to agiven antigen with high affinity, since different lymphocytes expressdifferent TCR co-receptors. Diversity of antigen presentation, mediatedby MHC classes I and II, is attained in at least three ways:

(1) an organism's MEW repertoire is usually polygenic (via multiple,interacting genes);

(2) MHC expression is codominant (from both sets of inherited alleles);and

(3) MHC gene variants are highly polymorphic (diversely varying fromorganism to organism within a species).

MHC molecules bind to both the T cell receptor and a CD4/CD8 co-receptoron T lymphocytes. The antigen epitope held in the peptide-binding grooveof the MHC molecule interacts with the variable Ig-Like domain of theTCR to trigger T-cell activation. However, the MEW molecules can alsothemselves act as antigens and can provoke an immune response in therecipient of a tissue or cells which express a foreign MHC, thus causingtransplant rejection. Still further, the transplantation ofimmunocompetent cells can actually result in rejection of host tissue,also known as graft vs host disease. In this regard, each human cellexpresses six MHC class I alleles (one HLA-A, -B, and -C allele fromeach parent) and six to eight MHC class II alleles (one HLA-DP and -DQ,and one or two HLA-DR from each parent, and combinations of these). TheMHC variation in the human population is high, with at least 350 allelesfor HLA-A genes, 620 alleles for HLA-B, 400 alleles for DR, and 90alleles for DQ. Any two individuals who are not identical twins willexpress differing MHC molecules.

All MHC molecules can mediate transplant rejection, but HLA-C andHLA-DP, which show low polymorphism, are less important. Transplantrejection can be minimised by attempting to match as much of the cellsurface HLA repertoire as possible between a donor and a recipient. Acomplete match is only possible as between identical twins. However,selecting donors based on minimising incompatibility at one or more ofthe range of HLA antigens expressed on a cell is highly desirable andcan significantly minimise rejection problems. This is a particularissue addressed by the present invention since the usual method ofmanaging tissue/cell rejection is the administration ofimmunosuppressive treatment regimes, this not being desirable in thecontext of a treatment regime based on the administration of geneticallymodified immune cells which are required to function at an optimum levelof functionality. In accordance with the present invention, this can beachieved by utilizing cells, such as iPSCs, or cells such as T cellsfrom which iPSCs are derived, which are homozygous for one or more MHChaplotypes, the HLA allele of interest being one which is a majortransplantation antigen and which is preferably expressed by asignificant proportion of the population, such as at least 5%, at least10%, at least 15%, at least 17%, at least 20%, or more of thepopulation. Where the homozygous HLA haplotype corresponds to a dominantMHC I or MHC II HLA type (in terms of tissue rejection), the use of sucha cell will result in significantly reduced problems with tissuerejection in the wider population who receive the cells of the presentinvention in the context of a treatment regime. In terms of the presentinvention, the genetically modified cells may be homozygous in relationto one cellular HLA antigen or they may be homozygous in relation tomore than one HLA antigen, e.g., 2, 3, or more HLA antigens. In someembodiments, the genetically modified cells are homozygous in relationto one HLA antigen selected from those listed in Table 1, includinge.g., HLA A1, B8, C7, DR17, DQ2, or HLA A2, B44, C5, DR4, DQ8, or HLAA3, B7, C7, DR15, DQ6. In some embodiments, the genetically modifiedcells are homozygous in relation to two or more HLA antigens selectedfrom those listed in Table 1, including e.g., HLA A1, B8, C7, DR17, DQ2,or HLA A2, B44, C5, DR4, DQ8, or HLA A3, B7, C7, DR15, DQ6.

The term “HLA-type” should therefore be understood to refer to thecomplement of HLA antigens present on the cells of an individual.

Obtaining a suitable homozygous HLA T cell for use in generating an iPSCcan be achieved by any suitable method including, for example, screeninga population (such as via a blood bank) to identify individualsexpressing HLA homozygocity and then screening for T cells from thatindividual which exhibit the TCR specificity of interest. These normallyvery rare T cells can be selectively stimulated by the specificantigenic peptide that their TCR recognises and vastly increased infrequency (e.g., from <0.0001 to 0.2).

It would be appreciated by the person skilled in the art thatsignificant information is widely available in the public literaturewhich describes the identification and utility of homozygous haplotypesin terms of minimizing donor-recipient HLA mismatch across a givenpopulation of interest, thereby enabling the generation of donor banks.See for example Pappas et al (2015). In one example, Table 1 identifiesthe 15 highest ranked homozygous HLA haplotypes relative to theproportion of the UK population to which this provides minimal mismatch.The first 8 listed homozygous HLA haplotypes are compatible with 49% ofthe population. A further example is outlined in Table 2 which detailsthe first 10 ranked haplotypes compatible with the ethnically diverseCalifornian population. Table 2 includes match frequencies forsubpopulations, including, black or African American, Asian and PacificIslander, white, Hispanic and American Indian and Alaska natives.Further still, Table 3 outlines the 50 most frequent haplotypes forHLA-A-B-DR, A-B, A-DR and B-DR in the North China population. It wouldbe appreciated that a person skilled in the art would understand thatthe data depicted in Table 3 can be used to define a set of homozygoushaplotypes which would provide minimal mismatch for the North Chinesepopulation.

TABLE 1 Utility of 15 highest ranked homozygous HLA-A, -B, -DR typesidentified to provide a zero HLA mismatch for the UK population.Recipients Recipients matched matched Rank HLA-A HLA-B HLA-DR (%)(cumulative %) 1 A1 B8 DR17(3) 16.87 16.87 2 A2 B44(12) DR4 9.51 26.38 3A3 B7 DR15(2) 7.45 33.83 4 A2 B7 DR15(2) 4.28 38.11 5 A2 B44(12) DR73.41 41.52 6 A2 B62(15) DR4 2.85 44.37 7 A1 B57(17) DR7 2.54 46.91 8 A3B35 DR1 2.10 49.01 9 A29(19) B44(12) DR7 2.04 51.05 10 A2 B60(40) DR41.75 52.80 11 A2 B8 DR17(3) 1.60 54.40 12 A2 B27 DR1 1.28 55.68 13 A2B44(12) DR13(6) 1.23 56.91 14 A3 B7 DR4 1.20 58.11 15 A1 B8 DR4 0.9459.05

TABLE 2 Top cis and trans matched haplolines of the Californiapopulation. CIS TRANS Haplotype HLA- Match Match Expected CIS matchfrequency A~B~DRB1 % (K_(i)) SD % (K_(i)) SD f_(CAU) f_(HIS) f_(API)f_(AFA) f_(NAM) f_(exp) 01:01g~08:01g~03:01 6.32 0.24 6.64 0.26 11.633.57 0.47 2.181 8.484 5.59 03:01g~07:02g~15:01 3.47 0.18 1.06 0.19 5.9672.37 0.4 1.198 4.618 3.06 29:02g~44:03~07:01 2.57 0.15 2.71 0.15 3.7311.17 0.11 0.68 2.88 1.8 02:01g~07:02g~15:01 2.03 0.15 3.6 0.2 3.565 0.760.06 0.927 3.206 1.64 02:01g~44:02g~04:01 1.85 0.13 2.22 0.15 2.851 3.630.07 0.779 2.55 2.23 01:01g~57:01g~07:01 1.68 0.13 1.95 0.14 2.356 0.840.35 0.465 1.617 1.2 03:01g~35:01g~01:01 1.35 0.11 1.61 0.11 2.521 0.470.05 0.435 1.877 1.11 02:01g~15:01g~04:01 1.24 0.12 1.57 0.14 2.124 0.892.89 0.43 1.973 1.47 30:01g~13:02g~07:01 1.24 0.12 1.3 0.12 1.663 0.30.04 0.282 1.203 0.73 33:01g~14:02~01:02 0.99 0.1 1.02 0.1 1.532 0.620.08 0.304 1.21 0.79 Abbreviations AFA, black or African American; API,Asian and Pacific Islander; CAU, white (non-Hispanic); CIS, cis matchbenefit; f_(exp), expected cis match frequency; HIS, Hispanic; K_(i),number of matches as a count or percentages of the total number ofsubjects; NAM, American Indian and Alaska native; TRANS, trans matchbenefit.

TABLE 3 50 most frequent haplotypes for HLA-A-B-DR, A-B, A-DR and B-DR(at 10⁻⁵) HF = haplotype frequency per 100,000 HLA-A-B-DR HLA-A-A-BHLA-A-DR HLA-A-B-DR Haplotype HF R.L.D Haplotype HF R.L.D Haplotype HFR.L.D Haplotype HF R.L.D A30-B13- 4446 0.58 A30-B13 5538 0.81 A2-DR95882 0.23 B13-DR7 5617 0.44 DR7 A2-B46- 2388 0.16 A2-B46 5090 0.60A2-DR15 4703 −0.04 B46-DR9 3225 0.37 DR9 A33-B58- 1436 0.29 A33-B58 32010.74 A30-DR7 4532 0.64 B13-DR12 2303 0.12 DR17 A2-B13- 1088 0.04 A2-B612592 0.17 A2-DR12 4118 0.13 B52-DR15 2285 0.55 DR12 A2-B46- 1046 0.07A2-B51 2411 0.6 A11-DR15 3426 0.03 B62-DR4 2070 0.18 DR8 A33-B58- 10100.18 A2-B62 2198 0.00 A24-DR15 3143 0.03 B61-DR9 2045 0.22 DR13 A33-B44-936 0.14 A11-B60 2197 0.18 A2-DR4 2987 −0.11 B62-DR15 1921 0.11 DR13A2-B61- 904 0.01 A2-B13 2136 −0.35 A11-DR12 2979 0.12 B44-DR7 1869 0.28DR9 A1-B37- 860 0.46 A11-B62 2106 0.13 A11-DR4 2704 0.07 B7-DR15 18670.29 DR10 A11-B75- 848 0.12 A2-B60 1879 −0.3 A2-DR8 2660 0.20 B58-DR171858 0.42 DR12 A11-B62- 814 0.04 A24-B61 1802 0.15 A24-DR4 2584 0.08B51-DR9 1727 0.12 DR4 A24-B54- 697 0.11 A24-B62 1798 0.10 A11-DR9 23780.01 B54-DR4 1497 0.38 DR4 A2-B62- 676 0.02 A24-B60 1765 0.13 A24-DR92375 0.03 B44-DR13 1480 0.25 DR15 A3-B7- 658 0.10 A33-B44 1752 0.29A2-DR14 2169 0.08 B46-DR8 1453 0.18 DR15 A1-B57- 647 0.38 A11-B13 1689−0.16 A33-DR13 2057 0.34 B75-DR12 1405 0.26 DR7 A11-B7- 647 0.11 A2-B751679 0.18 A2-DR11 2012 −0.01 B60-DR15 1325 0.04 DR1 A24-B61- 607 0.03A11-B75 1632 0.28 A24-DR12 1860 0.02 B58-DR13 1269 0.26 DR9 A2-B51- 5970.15 A24-B54 1468 0.33 A2-DR7 1553 −0.53 B13-DR15 1204 −0.35 DR9 A2-B61-586 0.02 A2-B35 1446 −0.19 A24-DR11 1524 0.07 B37-DR10 1188 0.67 DR12A24-B62- 579 0.02 A24-B51 1445 0.4 A33-DR17 1502 0.31 B35-DR15 1122 0.02DR4 A32-B52- 575 0.23 A11-B51 1399 0.1 A24-DR14 1410 0.08 B7-DR1 11020.24 DR15 A11-B13- 572 0.03 A2-B48 1365 0.18 A11-DR14 1348 0.04 B61-DR121094 0.07 DR15 A11-B62- 556 0.01 A1-B37 1325 0.69 A11-DR8 1211 0.02B8-DR17 1058 0.82 DR15 A33-B44- 532 0.05 A2-B38 1264 0.19 A3-DR15 11730.07 B61-DR15 1001 −0.07 DR7 A11-B13- 532 0.00 A24-B35 1079 0.3 A11-DR111070 −0.15 B57-DR7 986 0.65 DR12 A11-B52- 512 0.01 A11-B52 1060 0.13A1-DR7 1011 0.08 B75-DR15 986 0.09 DR15 A32-B44- 493 0.19 A24-B48 10030.17 A2-DR16 958 0.19 B51-DR15 982 −0.21 DR7 A2-B75- 484 0.03 A3-B7 10010.18 A24-DR8 954 −0.03 B60-DR9 967 0.02 DR9 A11-B51- 452 0.02 A1-B57 9840.67 A11-DR1 921 0.06 B35-DR11 949 0.10 DR9 A2-B13- 431 0.67 A3-B35 9600.13 A1-DR10 887 0.48 B60-DR4 943 0.03 DR7 A2-B46- 427 0.01 A11-B46 906−0.30 A31-DR15 879 0.08 B60-DR11 929 0.08 DR14 A11-B75- 425 0.02 A24-B13885 −0.50 A33-DR7 863 0.01 B62-DR12 850 0.01 DR15 A24-B60- 420 0.01A2-B54 874 −0.08 A3-DR1 785 0.15 B51-DR4 837 −0.01 DR15 A2-B60- 417 0.01A11-B7 848 0.1 A1-DR15 781 −0.13 B62-DR9 821 −0.15 DR15 A24-B51- 4140.01 A11-B35 787 −0.27 A2-DR17 691 −0.43 B51-DR11 794 0.04 DR9 A2-B62-408 0.30 A11-B61 776 −0.32 A3-DR7 691 0.02 B60-DR12 786 0.01 DR4 A2-B75-397 0.21 A32-B44 763 0.34 A32-DR15 683 0.20 B60-DR8 771 0.06 DR12A2-B46- 389 0.28 A31-B51 757 0.14 A3-DR4 678 0.02 B75-DR9 750 0.06 DR12A11-B46- 387 0.28 A29-B7 726 0.62 A11-DR7 652 −0.68 B51-DR12 701 −0.11DR9 A11-B60- 379 0.01 A3-B44 705 0.9 A2-DR13 624 −0.59 B27-DR4 693 0.25DR9 A11-B60- 378 0.03 A2-B71 697 0.23 A26-DR15 616 0.02 B48-DR15 6650.05 DR8 A24-B13- 377 0.01 A24-B7 679 −0.5 A2-DR1 609 −0.49 B71-DR4 6630.35 DR12 A2-B54- 377 0.09 A32-B52 677 0.31 A24-DR7 596 −0.67 B51-DR14644 0.03 DR4 A2-B75- 362 0.00 A11-B55 674 0.19 A32-DR7 579 0.19 B50-DR7640 0.68 DR15 A24-B7- 362 0.01 A2-B55 670 0.7 A1-DR13 572 0.06 B35-DR9632 −0.18 DR15 A2-B71- 350 0.10 A2-B39 654 0.7 A33-DR15 550 −0.54B35-DR4 625 −0.09 DR4 A11-B60- 350 0.19 A11-B54 649 0.2 A3-DR13 484 0.04B46-DR14 612 0.03 DR15 A2-B61- 345 0.15 A2-B67 620 0.56 A31-DR9 480−0.01 B62-DR14 599 0.02 DR15 A2-B50- 332 0.21 A24-B46 604 −0.46 A33-DR4476 −0.42 B48-DR9 556 0.05 DR7 A2-B48- 332 0.02 A31-B62 570 0.8 A26-DR4468 0.02 B35-DR1 542 0.08 DR9

As detailed hereinbefore, the present invention is predicated on thedetermination that a stem cell can be consistently and stably engineeredto express dual T cell and chimeric antigen receptors directed tomultiple distinct antigens, thereby providing an ongoing source of Tcells which are more therapeutically effective than the cells used incurrently available therapeutic cellular treatment regimes. In thisregard, reference to a “stem cell” should be understood as a referenceto any cell which exhibits the potentiality to develop in the directionof multiple lineages, given its particular genetic constitution, andthus to form a new organism or to regenerate a tissue or cellularpopulation of an organism. The stem cells which are utilised inaccordance with the present invention may be of any suitable typecapable of differentiating along two or more lineages and include, butare not limited to, embryonic stem cells, adult stem cells, umbilicalcord stem cells, haemopoietic stem cells (HSCs), totipotent cells,progenitor cells, precursor cells, pluripotent cells, multipotent cellsor de-differentiated somatic cells (such as an induced pluripotent stemcell). By “totipotent” is meant that the subject stem cell can selfrenew. By “pluripotent” is meant that the subject stem cell candifferentiate to form, inter alia, cells of any one of the three germlayers, these being the ectoderm, endoderm and mesoderm.

In one particular embodiment, the subject stem cell is an inducedpluripotent stem cell (iPSC). Without limiting the present invention toany one theory or mode of action, adult stem cell expansion is notnecessarily based on the occurrence of asymmetrical stem cell divisionin order to effect both stem cell renewal and differentiation along aspecific somatic cell lineage. In particular, pluripotent stem cells canbe sourced from T cells which are induced to transition to a state ofmultilineage potential. The development of technology to enable thede-differentiation of adult cells is of significant importance due tothe difficulty of otherwise inducing stem cell renewal and expansion invitro.

According to this embodiment there is therefore provided a geneticallymodified mammalian stem cell, or a T cell differentiated therefrom,which stem cell is an iPSC, is capable of differentiating to a T cellexpressing a TCR directed to a first antigenic determinant, andcomprises a nucleic acid molecule encoding a chimeric antigen receptor,wherein said receptor comprises an antigen recognition moiety directedto a second antigenic determinant, which antigen recognition moiety isoperably linked to a T cell activation moiety. In one embodiment, thegenetically modified mammalian iPSC expresses at least one homozygousHLA haplotype.

iPSCs are usually generated directly from somatic cells, although itshould be understood that the present invention is not limited in thisregard. That is, the subject iPSC may be generated from a cell which isnot terminally differentiated; indeed iPSC can be induced in principlefrom any nucleated cell including, for example, mononucleocytes fromblood and skin cells. For example, in the context of one embodiment ofthe present invention, the subject iPSCs may be generated from fullydifferentiated T cells or they may be generated from precursor T cells,such as thymocytes. To the extent that the subject thymocyte hasre-arranged its TCR and exhibits an antigen specificity of interest inthe context of the present invention, one may seek to generate the iPSCfrom this cell. This may be relevant, for example, where the particularTCR rearrangement in issue is one which might be expected to be selectedagainst during thymopoiesis. It would be appreciated by the skilledperson that one of the complicating factors with respect toimmunoresponsiveness to tumour cells or autoreactive cells is that inthis situation the immune system is required to direct an immuneresponse to a self cell and, therefore, a self-antigen. Such immunecells are usually selected against during T lymphocyte differentiationin the thymus in order to minimize the prospect of the onset of anautoimmune disease. In the context of neoplastic and autoimmuneconditions, however, the unwanted cell is a self cell and, accordingly,the cell surface antigens which one may seek to target will be selfantigens. Without limiting the present invention in any way, and asdiscussed in more detail hereafter, one of the advantages of using aniPSC from which to generate a TCR/CAR expressing T cell directed tomultiple distinct antigenic determinants is that it has been determinedthat the actions of epigenetic memory may potentiate the differentiationof an iPSC to a functional T cell which expresses a TCR directed to thesame antigen as the T cell from which the iPSC has been derived.However, in terms of selecting a specific TCR expressing cell from whichto derive an iPSC, it may be difficult to identify a suitable fullydifferentiated T cell since a T cell expressing a functional TCRdirected to a self antigen may have been selected against duringthymopoiesis. It may therefore be more feasible to screen for athymocyte which expresses the TCR re-arrangement of interest, whichthymocyte has not yet undergone negative selection to remove potentiallyself reactive cells.

In another embodiment, an iPSC is transfected with one or more nucleicacid molecules coding for a TCR (such as rearranged TCR genes) directedto a first antigenic determinant (e.g., a tumour antigenic determinant).

In still another embodiment, the subject stem cell is a haemopoieticstem cell (HSC). Haemopoietic stem cells (HSCs) refer to stem cells thatgive rise to all the blood cells of the lymphoid and myeloid lineagesthrough the process of haematopoiesis. HSCs are derived from mesoderm,and can be found in adult bone marrow, peripheral blood, and umbilicalcord blood. HSCs can be collected from bone marrow, peripheral blood,and umbilical cord blood by established techniques, and are commonlyassociated with CD34+ expression. In some embodiments, human HSCs can bedefined as being CD34+CD38−CD90+CD45RA− (see Reinisch et al (2015)). AnHSC can be genetically modified, e.g., transfected, with one or morenucleic acids encoding a TCR directed to a first antigenic determinant,then subsequently directed to differentiate into a T cell. Nucleic acidsencoding one or more CARs, and optionally nucleic acids encoding one ormore docking antigen-binding receptors, can also be introduced into anHSC, before or after differentiation of the HSC into a T cell.

Reference to a “T cell receptor” (TCR) should therefore be understood asa reference to the heterodimer found on the surface of T cells or NKTcells which recognise peptides presented by MHC. Specifically, CD8+ Tcells recognise peptide presented in the context of MHC class I whileCD4+ T cells recognise peptide presented in the context of WIC class II.Without limiting the present invention to any one theory or mode ofaction, in the majority of human T cells, the TCR comprises an α and βchain, while a minor population of cells express a TCR comprising a γδheterodimer. The TCR is a disulfide-linked membrane-anchoredheterodimeric protein. The γ, δ, α and β chains are composed of twoextracellular domains: a variable (V) region and a constant (C) region,which both form part of the immunoglobulin superfamily and which fold toform antiparallel β-sheets. The constant region is proximal to the cellmembrane, followed by a transmembrane region and a short cytoplasmictail, while the variable region binds to the peptide/WIC complex.

The variable domains of both the TCR α-chain and β-chain each expressthree hypervariable or complementarity determining regions (CDRs),whereas the variable region of the β-chain has an additional area ofhypervariability (HV4) that does not normally contact antigen and,therefore, is not considered a CDR. The processes for the generation ofTCR diversity are based mainly on genetic recombination of the DNAencoded segments in precursor T cells—either somatic V(D)J recombinationusing RAG1 and RAG2 recombinases or gene conversion using cytidinedeaminases. Each recombined TCR possesses unique antigen specificity,determined by the structure of the antigen-binding site formed by the αand β chains, in the case of αβ T cells, or γ and δ chains in the caseof γδ T cells. The TCR α chain is generated by VJ recombination, whereasthe β chain is generated by VDJ recombination. Likewise, generation ofthe TCR γ chain involves VJ recombination, whereas generation of the TCRδ chain occurs by VDJ recombination. The intersection of these specificregions (V and J for the α or γ chain; V, D, and J for the β and δchain) corresponds to the CDR3 region that is important for peptide/MHCrecognition. It is the unique combination of the segments at thisregion, along with palindromic and random nucleotide additions, whichaccount for the even greater diversity of T cell receptor specificityfor processed antigenic peptides.

Accordingly, reference to a TCR “directed” to an antigenic determinantshould be understood as a reference to a TCR which has undergonerearrangement and which exhibits specificity for an antigenicdeterminant, preferably a self (particularly a self cancer) antigenicdeterminant.

In one embodiment, an iPSC is derived from a cell which expresses arearranged TCR, preferably a rearranged αβ TCR. Examples of cellssuitable for use in generating the iPSCs of the present inventioninclude, but are not limited to CD4⁺ T cells, CD8⁺ T cells, NKT cells,thymocytes or other form of precursor T cells. In another embodiment,said cell expresses a rearranged γδ TCR.

There is therefore provided a genetically modified mammalian iPSC orHSC, or a T cell differentiated therefrom, which iPSC or HSC is capableof differentiating to a T cell expressing a TCR directed to a firstantigenic determinant, is derived from a cell in which the TCR geneshave undergone re-arrangement, or has been transduced with saidrearranged genes, and comprises a nucleic acid molecule encoding achimeric antigen receptor, wherein said receptor comprises an antigenrecognition moiety directed to a second antigenic determinant, whichantigen recognition moiety is operably linked to a T cell activationmoiety. In some embodiments, the genetically modified mammalian iPSC orHSC expresses at least one homozygous HLA haplotype.

In one embodiment, said iPSC is derived from a T cell or a thymocyte.

In another embodiment, said iPSC is derived from a T cell or thymocyteexpressing an αβ TCR.

In still another embodiment, said iPSC is derived from a T cell orthymocyte expressing a γδ TCR.

The subject stem cells may have been freshly isolated from an individualwho is the subject of treatment or they may have been sourced from anon-fresh source, such as from a culture (for example, where cellnumbers were expanded and/or the cells were cultured so as to renderthem receptive to differentiation signals) or a frozen stock of cells,which had been isolated at some earlier time point either from anindividual or from another source. It should also be understood that thesubject cells, prior to undergoing differentiation, may have undergonesome other form of treatment or manipulation, such as but not limited topurification, modification of cell cycle status or the formation of acell line such as an embryonic stem cell line. Accordingly, the subjectcell may be a primary cell or a secondary cell. A primary cell is onewhich has been isolated from an individual. A secondary cell is onewhich, following its isolation, has undergone some form of in vitromanipulation such as the preparation of an embryonic stem cell line,prior to the application of the method of the invention.

To the extent that the stem cells of the present invention are iPSCs,methods for generating iPSCs are well known to the person of skill inthe art. In this regard, and as detailed hereinbefore, iPSCs are cellswhich are derived from a more mature cell type, such as a somatic cell,which has been transitioned/de-differentiated back to a pluripotentstate.

Without limiting the present invention to any one theory or mode ofaction, iPSCs can be derived by introducing a specific set ofpluripotency-associated genes, or “reprogramming factors”, into asomatic cell type. The most commonly used set of reprogramming factors(also know as the Yamanaka factors) are the genes Oct4 (Pou5f1), Sox2,cMyc, and Klf4. The transfection of these four specific genes encodingtranscription factors were shown by Yamanaka in 2006 to convert adulthuman cells into pluripotent cells. While this combination is the mostconventional combination used for producing iPSCs, each of the factorscan be functionally replaced by related transcription factors, miRNAs,small molecules, or even non-related genes such as lineage specifiers.For example, the induction of iPSCs following transfection of Oct 3/4,Sox2, Klf4 and c-Myc using a retroviral system has been achieved, as ithas also been via the transfection of Oct4, Sox2, Nanog and Lin28 usinga lentiviral system. The former set of transcription factors are knownas the Yamanaka factors while the latter are commonly known as theThomson factors. As would be appreciated by the person of skill in theart, a wide range of modifications to the basic reprogramming factorexpression vectors have been made and new modes of delivery have beendesigned in order to increase efficiency and minimise or remove vectorsequences that might otherwise be integrated into the reprogrammed iPSCgenome. These methods would be well known to the skilled person andinclude, but are not limited to:

single cassette reprogramming vectors with Cre-Lox mediated transgeneexcision;

(ii) reprogramming by non-integrating viruses such as adenovirus orsendai virus.

Alternatively, expression of reprogramming factors as proteins providesa means of generating iPSCs which have not undergone integration of theintroduced vector DNA into the germline.

Non-viral reprograming methods have also been developed. These include,but are not limited to:

mRNA Transfection—The ability to express reprogramming factors as mRNAoffers a method to make iPSCs into which chromosomal integration ofviral vectors does not occur. Warren et al. transcribes mRNAs toefficiently express reprogramming factors (Warren et al (2010)). Byadding Lin28 to the Yamanaka reprogramming factor protocol, culturing at5% 02, and including valproic acid in the cell culture medium, theefficiency can be increased. Reprogramming factor mRNAs are commerciallyavailable.

(ii) miRNA Infection/Transfection—Several miRNA clusters are stronglyexpressed in embryonic stem cells. When synthetic mimics of the maturemiR-302b and/or miR-372 plus the four lentiviral Yamanaka factors areadded to MRCS and BJ-1 fibroblasts there is a 10- to 15-fold increase inreprogramming efficiency in comparison with the four lentiviral factorsalone (Subramanyam et al (2011)). It has also been found that certainmiRNAs can reprogram cells at high efficiency without the presence ofthe Yamanaka factors.

(iii) PiggyBac—PiggyBac is a mobile genetic element (transposon) that inthe presence of a transposase can be integrated into chromosomal TTAAsites and subsequently excised from the genome upon re-expression of thetransposase. When cloned into a piggyBac vector and co-transfected intoMEFs the Yamanaka factors can reprogram cells 14-25 dayspost-transfection (Kaji et al (2009); Woltj en et al (2009)). ThepiggyBac vector can be excised from the iPSCs upon re-expression of thetransposase.

(iv) Minicircle Vectors—Minicircle vectors are minimal vectorscontaining only the eukaryotic promoter and cDNA(s) that will beexpressed. A Lin28, GFP, Nanog, Sox2, and Oct4 minicircle vectorexpressed in human adipose stromal cells is able to reprogram cells(Narsinh et al (2011)).

(v) Episomal Plasmids—Transient expression of reprogramming factors asepisomal plasmids allows for the generation of iPSCs. For exampleoriP/EBNA vectors can be constructed with the Yamanaka factors plusLin28 in one cassette and another oriP/EBNA vector containing SV40 largeT antigen (Chuo et al (2011)). These vectors have been shown to beexpressed in CD34+ cord blood, peripheral blood, and bone mononuclearcells in media supplemented with sodium butyrate, resulting in iPSCcolonies in 14 days. The transfected plasmids are ultimately lost.

In another aspect, the skilled person would also be familiar withadjunct methods which are known to enhance the programming efficiency ofcells. For example, even when using the same method there can bevariability in iPSC efficiency between cells. Various small moleculeshave been shown to enhance reprogramming efficiency (Table 4).

TABLE 4 Compounds increasing iPSC reprogramming efficiency TreatmentProcess affected Valproic acid Histone deacetylase inhibition Sodiumbutyrate Histone deacetylase inhibition PD0325901 MEK inhibition A-83-01TGFβ-inhibition SB43152 TGFβ-inhibition Vitamin C Enhances epigeneticmodifiers, promotes survival of antioxidant effects Thiazovivin ROCKinhibitor, promotes cell survival PS48 P13K/Akit activation, promotesglycolysis 5% Oxygen Promotes glycolysis

Several known mechanisms enable these molecules to facilitatereprogramming including inhibition of histone deacetylation (Mali et al(2010); Huangfu et al (2008)) blockade of the TGFβ and MEK signallingpathways (Lin et al (2009); Ichida et al (2009)), enhancement offunction of epigenetic modifiers (Esteban et al (2010)), inhibition ofthe ROCK pathway (Noggle et al (2011)) and induction of glycolysis (Zhuet al (2010)). Amongst these small molecules, the histone deacetylataseinhibitors valproic acid and sodium butyrate are the most commonly usedin reprogramming protocols. It should also be noted that culture ofcells in 5% oxygen during the reprogramming process can also increaseefficiency of iPSC derivation (Yoshida et al (2009)). For cells that areparticularly difficult to reprogram, the addition of a small moleculeand culture in hypoxic conditions can yield improvements. Another optionis to use embryonic stem cell-conditioned medium (ESCM) to induceexpression of endogenous reprogramming factors (Balasubramanian et al(2009)). The efficiency can be improved further with the addition ofvalproic acid. Such a strategy can also be used to enhance the abilityof exogenously introduced reprogramming factors to increasereprogramming efficiency.

To the extent that the stem cells of the present invention are HSCs,methods for generating or preparing HSCs are well known to the person ofskill in the art. HSCs can be obtained by direct extraction from thebone marrow or from the blood after the HSCs are released from the bonemarrow following e.g., treatment with specific molecules such as GM-CSF.The HSCs can then be purified through their plasma membrane expressionof CD34 by for example magnetic beads coated with anti-CD34 or cellsorting by flow cytometry after labelling with fluorescent anti CD34.These so purified HSCs can be induced to T cell differentiation usingthe OP 9/OP9 DL-L1 system outlined in Example 3 and FIGS. 3-10inclusive.

Reference to the subject stem cell, in particular iPSC or HSC, being“capable of” differentiating to a T cell expressing a TCR directed to anantigenic determinant should be understood as a reference to a cellwhich either does, or has the capacity to, transcribe and translate thesubject TCR genes and then assemble the TCR heterodimer as a functionalreceptor on the cell surface. As would be appreciated by the skilledperson, in most situations a stem cell such as an iPSC will not, in itsundifferentiated form, express a TCR. TCR expression is generallyexpected to occur once directed differentiation along the T cell lineagehas been induced. In one embodiment, the cell is one which, with orwithout a CAR genetic modification, can be induced to differentiate to aT cell expressing a functional TCR. It should be understood that thecapacity of the cell to express a TCR of a particular specificity may beenabled by any suitable means. For example, the cell may have beentransfected with genes encoding the two TCR chains (eg. α and β chain)which, when expressed, will associate to form the TCR heterodimer.Alternatively, and in the context of a preferred embodiment of thepresent invention, the stem cell of the present invention is one whichhas been generated from a T cell, thymocyte or other cell in which theTCR genes have been rearranged. It has been determined that an iPSCwhich has been generated from such a cell, if directed to differentiateto a CD4⁺ or CD8⁺ T cell under appropriate cell culture conditions, willexpress the same TCR antigen specificity as the somatic T cell fromwhich the iPSC was derived. Of still further significance, and asdiscussed in more detail hereinafter, is that it has been determinedthat with or without transfection of the iPSC or HSC with one or morenucleic acids encoding one or more CARs, or the α and β chains of anantigen/MHC class I specific TCR, the T cell differentiated therefrom iscapable of stably expressing both a functional TCR and one or more CARs(and optionally one or more antigen-binding receptors), and is thereforedirected to two or more distinct antigenic determinants. Accordingly,such a stem cell is deemed “capable of” differentiating to a T cell andexpressing the requisite TCR on the basis that if the iPSC or HSC isprovided with the appropriate differentiative signal, this will occur.In this regard, since the rearrangement of the TCR genes is an entirelyindependent genomic event, the choice of T cell sub-population fromwhich to generate the iPSC need not necessarily be the same as the Tcell sub-population which it is sought to ultimately be produced via thedirected differentiation of the iPSC. For example, one may select a CD4⁺T cell which exhibits an appropriate TCR specificity in order togenerate an iPSC. However, once that iPSC has been generated, theskilled person may seek to direct the differentiation of the iPSC to aCD8⁺ T cell. In this case, by virtue of epigenetic memory, the newlygenerated CD8⁺ T cell will exhibit the functionality of a CD8⁺ T cellbut the TCR specificity will be that of the CD4⁺ T cell from which theiPSC was derived. The converse is also true.

Reference to inducing the “transition” of a somatic cell, such as a Tcell, to a multilineage potential phenotype, such as an iPSC, should beunderstood as a reference to inducing the genetic, morphologic and/orfunctional changes which are required to change a somatic phenotype to amultilineage (pluripotent) phenotype of the type defined herein.

To the extent that one may elect to render an iPSC capable of producinga TCR via the transfection of the cell with DNA encoding a TCR, it wouldbe appreciated that this transfection may occur at any time point, suchas prior to the generation of the iPSC of the present invention,subsequently to the generation of the iPSC, or it may occursimultaneously with the CAR transfection.

As detailed hereinbefore, a somatic cell, in particular a T cell orthymocyte, can be induced to transition into a stem cell, that is afunctional state of multilineage differentiation potential. Accordingly,reference to a cell exhibiting “multilineage differentiation potential”or “multilineage potential” should be understood as a reference to acell which exhibits the potentiality to develop along more than onesomatic differentiative path. For example, the cell may be capable ofgenerating a limited range of somatic cell types, such cells usuallybeing referred to as pluripotent or multipotent. These cells exhibit thepotential to commit to a more limited range of lineages than atotipotent cell, the latter being a cell which can develop in any of thedifferentiation directions inherently possible including all the somaticlineages and the gametes.

Cells that are classically termed “progenitor” cells or “precursor”cells fall within the scope of the definition of “multilineagedifferentiation potential” on the basis that, under appropriatestimulatory conditions, they can give rise to cells of more than onesomatic lineage. To the extent that reference to “stem cell” is madeherein in terms of the cells generated by the method of the invention,this should be understood as a reference to a cell exhibitingmultilineage differentiative potential as herein defined.

In terms of the present invention, it should be understood that theimportant feature of the subject stem cell is that the multilineagedifferentiative potential which the cell exhibits includes the capacityto differentiate to a T cell and to express a TCR exhibiting specificityfor an antigen of interest. Whether the TCR specificity is inducedbefore or after the stem cell is generated (such as via the transfectionof the stem cell with DNA encoding the TCR of interest) is irrelevant.It should be understood that the stem cells claimed herein encompass allstem cells exhibiting the requisite differentiative potential,irrespective of when or how that capability has been introduced. Stillfurther, it should also be understood that the subject stem cells neednot be totipotent. Provided that they exhibit the capacity todifferentiate along more than one somatic cell lineage and provided thatone of these lineages is a T cell lineage, said cells fall within thescope of the present invention.

As detailed hereinbefore, the stem cells provided by the presentinvention are genetically modified. By “genetically modified” is meantthat the subject cell results from some form of molecular manipulationrelative to that which is observed in the context of a correspondingunmodified cell. In the context of the present invention, the subjectstem cell comprises a nucleic acid molecule encoding a chimeric antigenreceptor, and optionally further comprises a nucleic acid moleculeencoding an antigen-binding receptor. As disclosed herein, a nucleicacid encoding a receptor, whether a chimeric antigen receptor or anantigen-binding receptor, can be introduced to a stem cell such as aniPSC or an HSC, or to a cell (e.g., a T cell) from which a stem cell isderived; and in both instances, the resulting stem cell which comprisesthe receptor-encoding nucleic acid is considered herein to be agenetically modified stem cell. A T cell differentiated from agenetically modified stem cell, and a T cell engineered to contain anucleic acid encoding a genetically engineered CAR or antigen-bindingreceptor, are also considered herein genetically modified T cells.

Reference to a “nucleic acid molecule” should be understood as areference to both deoxyribonucleic acid and ribonucleic acid thereof.The subject nucleic acid molecule may be any suitable form of nucleicacid molecule including, for example, a genomic, cDNA or ribonucleicacid molecule. To this end, the term “expression” refers to thetranscription and translation of DNA or the translation of RNA resultingin the synthesis of a peptide, polypeptide or protein. A DNA construct,for example, corresponds to the construct which one may seek totransfect into a cell for subsequent expression while an example of anRNA construct is the RNA molecule transcribed from a DNA construct,which RNA construct merely requires translation to generate the proteinof interest. Reference to “expression product” is a reference to theproduct produced from the transcription and translation of a nucleicacid molecule.

Reference to “chimeric antigen receptor” (also known as an “artificial Tcell receptor”, “chimeric T cell receptor” and “chimericimmunoreceptors”) should be understood as a reference to engineeredreceptors which graft an antigen binding moiety onto an immune effectorcell. Typically, these receptors are used to graft the specificity of amonoclonal antibody onto a T cell; with transfection of their codingsequence facilitated by retroviral vectors. More specifically, andwithout limiting the invention in any way, the most common form of thesemolecules are fusions of single-chain variable fragments (scFv) derivedfrom monoclonal antibodies, fused to a CD3-zeta chain transmembrane andendodomain. Such molecules result in the transmission of a CD3-zetachain signal in response to recognition by the scFv of its target. WhenT cells express this chimeric molecule, they recognize and kill targetcells that express the antigen to which the scFv is directed. Forexample, to target malignant B cells, the specificity of T cells hasbeen redirected using a chimeric immunoreceptor specific for theB-lineage molecule, CD19.

The variable portions of an immunoglobulin heavy and light chain aregenerally fused by a flexible linker to form a scFv. This scFv isusually preceded by a signal peptide to direct the nascent protein tothe endoplasmic reticulum and subsequent surface expression, which thesignal peptide ultimately being cleaved. A flexible spacer allows thescFv to orient in different directions to enable antigen binding. Thetransmembrane domain is generally a typical hydrophobic alpha helixusually derived from the original molecule of the signalling endodomainwhich protrudes into the cell and transmits the desired signal.Accordingly, reference to an “antigen recognition moiety” should beunderstood as a reference to an extracellular portion of the receptorwhich recognises and binds to an antigenic determinant of interest, thatis, a target specific binding element. The antigen recognition domain isusually an scFv. There are, however, many other alternatives. Forexample, an antigen recognition moiety from native T-cell receptor (TCR)alpha and beta single chains have also been used, as have simpleectodomains (e.g., CD4 ectodomain to recognize HIV infected cells) andother recognition components such as a linked cytokine (which leads torecognition of cells bearing the cytokine receptor). In fact any moietythat binds a given target with sufficiently high affinity can be used asan antigen recognition domain. Such molecules are well known to theperson of skill in the art and selecting an appropriate molecule for usewould be well within the skill of the person in the art. In terms ofdesigning a chimeric antigen receptor, in particular the extracellulardomain, the skilled person may include additional moieties which areuseful in terms of effecting efficient expression or functioning. Forexample, and as detailed earlier, the nucleic acid molecule expressing aCAR may be designed to express a signal peptide at the N-terminal end ofthe antigen recognition moiety. Without limiting the present inventionto any one theory or mode of action, a signal peptide directs thenascent protein into the endoplasmic reticulum. This is necessary if thereceptor is to be glycosylated and anchored in the cell membrane. Anyeukaryotic signal peptide sequence may be used. Generally, a signalpeptide natively attached to the amino-terminal is used (e.g., in a scFvwith orientation light chain-linker-heavy chain, the native signal ofthe light-chain is used). In another example the extracellular domainmay also comprise a spacer region which may be used to link the antigenrecognition domain to the transmembrane domain. It should be flexibleenough to allow the antigen recognition domain to orient in differentdirections to facilitate antigen recognition and binding. The simplestform of a spacer region is the hinge region from IgG1. Alternativesinclude the CH₂CH₃ region of immunoglobulin and portions of CD3. Formost scFv based constructs, the IgG1 hinge suffices. Accordingly, theterm “spacer” refers to any oligo- or polypeptide that functions to linkthe transmembrane domain to either the extracellular domain or, thecytoplasmic domain in the polypeptide chain. A spacer domain maycomprise up to 300 amino acids, preferably 10 to 100 amino acids andmost preferably 25 to 50 amino acids. In yet another example, one maymodify the hinge region to change its length and thereby achieveadditional functional benefits. For example, in a traditional CAR whichcomprises a CD8 or CD28 hinge, a single Cysteine (Cys) can be left inthe hinge to stabilize dimerization on the T-cell surface. Thus two scFvare usually displayed (bivalent). In another example, one may substitutethe Cys (for Ser) so that the stabilizing disulphide bond cannot formthereby preventing dimerization and hence premature activation. The Cysmay also be removed entirely. Another design is to display just the VHdomain on one CAR and VL domain on another, thus the Cys pairing willalign the VH/VL to form a functional monovalent Fv, targeting theantigen of interest.

The antigen recognition moiety of the subject chimeric antigen receptoris operably linked to a T cell activation moiety. By “T cell activationmoiety” is meant the sub-region of the receptor which, after antigenrecognition and binding, is responsible for transmitting the signal intothe T cell to enable its activation and effector mechanism induction.The T cell activation moiety of a CAR is generally located within theintracellular domain (or “endodomain”) of the CAR; hence, theintracellular domain of a CAR molecule also typically comprises, or is,its “intracellular signalling domain”. A commonly used endodomaincomponent is the intracellular domain of CD3-zeta which contains 3ITAMs. This transmits an activation signal to the T cell after antigenis bound. CD3-zeta may not provide a fully competent activation signaland additional co-stimulatory signalling is desirable. For example,chimeric CD28 and OX40 can be used with CD3-Zeta to transmit aproliferative/survival signal, or all three can be used together. Itshould be understood that this intracellular signalling domain of theCAR is responsible for activation of at least one of the normal effectorfunctions of the immune cell, preferably a T cell in which the CAR hasbeen expressed. The term “intracellular signalling domain” refers to theportion of the protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signalling domain can be employed, in many cases itis not necessary to use the entire domain. To the extent that atruncated portion of the intracellular signalling domain is used, suchtruncated portion may be used in place of the intact chain as long as ittransduces the effector function signal. The term “intracellularsignalling domain” is thus meant to include any truncated portion of theintracellular domain sufficient to transduce the effector functionsignal.

Preferred examples of intracellular signalling domains for use in a CARinclude the cytoplasmic sequences of the T cell receptor (TCR) andco-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any synthetic sequence that has the samefunctional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signallingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signalling sequences) and thosethat act in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signalling sequences).Primary cytoplasmic signalling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signalling sequences that act in a stimulatorymanner may contain signalling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Examples of ITAM containingprimary cytoplasmic signalling sequences that are of particular useinclude those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It isparticularly preferred that cytoplasmic signalling molecule in the CARcomprise a cytoplasmic signalling sequence derived from CD3-zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR can bedesigned to comprise the CD3-zeta signalling domain by itself orcombined with any other desired cytoplasmic domain(s) useful in thecontext of the CAR of the invention. For example, the cytoplasmic domainof the CAR can comprise a CD3 zeta chain portion and a costimulatorysignalling region. The costimulatory signalling region refers to aportion of the CAR comprising the intracellular domain of acostimulatory molecule. A costimulatory molecule is a cell surfacemolecule other than an antigen receptor or its ligands that is requiredfor an efficient response of lymphocytes to an antigen. Examples of suchmolecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83, and thelike. The cytoplasmic signalling sequences within the cytoplasmicsignalling portion of the CAR of the invention may be linked to eachother in a random or specified order. Optionally, a short oligo- orpolypeptide linker, preferably between 2 and 10 amino acids in lengthmay form the linkage. A glycine-serine doublet provides a particularlysuitable linker. In one embodiment, the cytoplasmic domain is designedto comprise the signalling domain of CD3-zeta and the signalling domainof CD28.

As detailed hereinbefore, the antigen recognition moiety is operablylinked to the T cell activation moiety. By “operably linked” is meantthat the antigen recognition moiety is linked, bound or otherwiseassociated with the T cell activation moiety, such that upon binding ofthe antigen recognition moiety to the antigenic determinant, a signal isinduced via the T cell activation moiety to activate the subject T celland enable its effector functions to be activated. This is achieved, forexample, via the design of a transmembrane domain.

In one embodiment, the transmembrane domain that is naturally associatedwith one of the domains in the CAR is used. In some instances, thetransmembrane domain can be selected or modified by amino acidsubstitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex. Thetransmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. For example,transmembrane regions may be derived from (ie. comprise at least thetransmembrane region(s) of) the alpha, beta or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulinsuch as IgG4. Alternatively, the transmembrane domain may be synthetic,in which case it will comprise predominantly hydrophobic residues suchas leucine and valine. Preferably a triplet of phenylalanine, tryptophanand valine will be found at each end of a synthetic transmembranedomain. Optionally, a short oligo- or polypeptide linker, preferablybetween 2 and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signalling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.Typically, the transmembrane domain is a hydrophobic alpha helix thatspans the membrane. Generally, the transmembrane domain from the mostmembrane proximal component of the endodomain is used.

Reference to an “antigen-binding receptor” should be understood as areference to engineered receptors which are anchored to the cell surfaceand bind to an antigen. Similar to chimeric antigen receptors disclosedherein, antigen-binding receptors disclosed herein also comprise anantigen recognition moiety directed to an antigenic determinant. Theantigen recognition moiety in an antigen-binding receptor can take thesame form and designed in the same way as the antigen recognition moietyof a chimeric antigen receptor, as described herein. Also similar tochimeric antigen receptors disclosed herein, the antigenic recognitionmoiety in an antigen-binding receptor is operably linked (e.g., througha spacer sequence such as a hinge region) to a transmembrane domain,such that the antigen-binding receptor is anchored to the cell surface.The spacer sequence and the transmembrane domain in an antigen-bindingreceptor can also be designed in the same way as the spacer sequence andthe transmembrane domain of a chimeric antigen receptor, as describedabove. However, unlike chimeric antigen receptors, the antigen-bindingreceptor as defined herein is generally non-signalling, and may includean intracellular sequence that lacks a T-cell activation domain. Such anon-signalling antigen-binding receptor can bind to an antigen but doesnot trigger any signal transduction in T cells, and therefore is alsoreferred to as a “docking receptor” or “anchoring receptor”. Certainembodiments of antigen-binding receptors, such as a non-signallingCD47-binding receptor, are further described herein below.

Examples of nucleic acid constructs encoding a CAR and/or anantigen-binding receptor are depicted in FIG. 11, and exemplarysequences for CAR and antigen-binding receptor as well as variousdomains suitable for use in CARs and/or an antigen-binding receptors areprovided in SEQ ID NOS: 1-20.

It would be appreciated by the person of skill in the art that themechanism by which these genetic modifications are introduced into thecell may take any suitable form which would be well known and understoodby those of skill in the art. For example, genetic material is generallyconveniently introduced to cells via the use of an expression construct.

In one embodiment, a cell capable of differentiating into a T cellexpressing a TCR (i.e., a stem cell such as an iPSC or HSC) or a cellthat expresses a TCR from which a stem cell such as an iPSC can bederived, is transfected with a CAR-encoding expression construct. Theexpression construct can comprise one or more DNA regions comprising apromoter operably linked to a nucleotide sequence encoding a CAR and,optionally, a second DNA region encoding a selectable marker and,optionally, a third DNA region encoding a suicide protein. In thisregard, it should be appreciated that one may design the construct withany one or more additional components, such as a suicide gene, which theperson of skill in the art would deem useful, as a matter of routineprocedure. In the context of the cells of the present invention, whichare proposed to be used in vivo to treat patients, the ability tocontrol the killing of the genetically modified cells of the invention,and therefore effect their elimination from the in vivo environment, ishighly desirable. Without limiting the present invention to any onetheory or mode of action, the adoptive transfer of the cells of thepresent invention, particularly to the extent that they may be directedto “self” antigens such as tumour antigens or antigens expressed onautoreactive cells, or antigens to which cross-reactivity with selfantigens may occur, is not without risk. In this situation, outcomessimilar to graft versus host disease may occur, where these cells attackhealthy (non-diseased) cells. In the overall therapeutic scheme, theseside-effects may still be more desirable than the non-specific systemickilling of healthy tissue which is characteristic of a treatment such aschemotherapy or the uncontrolled killing of healthy tissue in anautoimmune disorder. Nevertheless, killing the cancer cells is paramountbut the ability to control the elimination of the cells of the presentinvention is highly desirable and can be routinely achieved by the verywell known and widely used technique of building an inducible suicidegene into the gene construct which is introduced into the stem/T cellsof the present invention.

The subject promoter may be constitutive or inducible. Where the subjectconstruct expresses more than one protein of interest, these may beunder the control of separate promoters or they may be under the controlof a single promoter, such as occurs in the context of a bicistronicvector which makes use of an IRES sequence to facilitate the translationof more than one protein product, in an unfused form, from a single RNAtranscript. The subject construct may additionally be designed tofacilitate use of the Cre recombinase mediated splicing inducible geneexpression system.

Reference to a nucleic acid “expression construct” should be understoodas a reference to a nucleic acid molecule which is transmissible to acell and designed to undergo transcription. The RNA molecule is thentranscribed therefrom. In general, expression constructs are alsoreferred to by a number of alternative terms, which terms are widelyutilised interchangeably, including “expression cassette” and “vector”.

For purposes of introducing nucleic acids encoding multiple receptors,whether the receptor is a CAR, an antigen-binding receptor, or acombination thereof, the multiple receptor-encoding nucleic acids can beplaced in one construct which is transfected into a cell. In oneembodiment, the multiple receptor-encoding nucleic acids can be includedin a multicistronic vector which makes use of an IRES sequence tofacilitate the translation of the multiple receptor proteins. In anotherembodiment, the multiple receptor-encoding nucleic acids can be linkedto each other within one expression unit and reading frame, for example,by utilizing a self-cleaving peptide (e.g., P2A) such that one singlepolypeptide comprising multiple receptor sequences is initially producedand subsequently processed to produce multiple receptors. In anotherembodiment, the multiple receptor-encoding nucleic acids are placed inseparate constructs which are used in transfection.

The expression construct of the present invention may be generated byany suitable method including recombinant or synthetic techniques. Tothis end, the subject construct may be constructed from firstprinciples, as would occur where an entirely synthetic approach isutilised, or it may be constructed by appropriately modifying anexisting vector. Where one adopts the latter approach, the range ofvectors which could be utilised as a starting point are extensive andinclude, but are not limited to:

-   -   (i) Plasmids: Plasmids are small independently replicating        pieces of cytoplasmic DNA, generally found in prokaryotic cells,        which are capable of autonomous replication. Plasmids are        commonly used in the context of molecular cloning due to their        capacity to be transferred from one organism to another. Without        limiting the present invention to any one theory or mode of        action, plasmids can remain episomal or they can become        incorporated into the genome of a host. Examples of plasmids        which one might utilise include the bacterial derived pBR322 and        pUC.    -   (ii) Bacteriophage: Bacteriophages are viruses which infect and        replicate in bacteria. They generally consist of a core of        nucleic acid enclosed within a protein coat (termed the capsid).        Depending on the type of phage, the nucleic acid may be either        DNA (single or double stranded) or RNA (single stranded) and        they may be either linear or circular. Phages may be        filamentous, polyhedral or polyhedral and tailed, the tubular        tails to which one or more tubular tail fibres are attached.        Phages can generally accommodate larger fragments of foreign DNA        than, for example, plasmids. Examples of phages include, but are        not limited to the E. coli lambda phages, P1 bacteriophage and        the T-even phages (eg. T4).    -   (iii) Baculovirus: These are any of a group of DNA viruses which        multiply only in invertebrates and are generally classified in        the family Baculoviridae. Their genome consists of        double-stranded circular DNA.    -   (iv) Mammalian virus: Examples of such viruses which infect        mammals, include lentivirus, sendai virus, retrovirus, and        vaccinia virus.    -   (v) Artificial Chromosomes: Artificial chromosomes such as yeast        artificial chromosomes or bacterial artificial chromosomes.    -   (vi) Hybrid vectors such as cosmids, phagemids and phasmids:        Cosmids are generally derived from plasmids but also comprise        cos sites for lambda phage while phagemids represent a chimeric        phage-plasmid vector. Phasmids generally also represent a        plasmid-phage chimaera but are defined by virtue of the fact        that they contain functional origins of replication of both.        Phasmids can therefore be propagated either as a plasmid or a        phage in an appropriate host strain.    -   (vii) Commercially available vectors which are themselves        entirely synthetically generated or are modified versions of        naturally occurring vectors, such as viral vectors.

It would be understood by the person of skill in the art that theselection of an appropriate vector for modification, to the extent thatone chooses to do this rather than synthetically generate a construct,will depend on a number of factors including the ultimate use to whichthe genetically modified cell will be put. For example, where the cellis to be administered in vivo into a human, it may be less desirable toutilise certain types of vectors, such as viral vectors. Further, it isnecessary to consider the amount of DNA which is sought to be introducedto the construct. It is generally understood that certain vectors aremore readily transfected into certain cell types. For example, the rangeof cell types which can act as a host for a given plasmid may vary fromone plasmid type to another. In still yet another example, the largerthe DNA insert which is required to be inserted, the more limited thechoice of vector from which the expression construct of the presentinvention is generated. To this end, the size of the inserted DNA canvary depending on factors such as the size of the DNA sequence encodingthe protein of interest, the number of proteins which are sought to beexpressed, the number of selection markers which are utilised and theincorporation of features such as linearisation polylinker regions andthe like.

The expression construct which is used in the present invention may beof any form including circular or linear. In this context, a “circular”nucleotide sequence should be understood as a reference to the circularnucleotide sequence portion of any nucleotide molecule. For example, thenucleotide sequence may be completely circular, such as a plasmid, or itmay be partly circular, such as the circular portion of a nucleotidemolecule generated during rolling circle replication (this may berelevant, for example, where a construct is being initially replicated,prior to its introduction to a cell population, by this type of methodrather than via a cellular based cloning system). In this context, the“circular” nucleotide sequence corresponds to the circular portion ofthis molecule. A “linear” nucleotide sequence should be understood as areference to any nucleotide sequence which is in essentially linearform. The linear sequence may be a linear nucleotide molecule or it maybe a linear portion of a nucleotide molecule which also comprises anon-linear portion such as a circular portion. An example of a linearnucleotide sequence includes, but is not limited to, a plasmid derivedconstruct which has been linearised in order to facilitate itsintegration into the chromosomes of a host cell or a construct which hasbeen synthetically generated in linear form. To this end, it should alsobe understood that the configuration of the construct of the presentinvention may or may not remain constant. For example, a circularplasmid-derived construct may be transfected into a cell where itremains a stable circular episome which undergoes replication andtranscription in this form. However, in another example, the subjectconstruct may be one which is transfected into a cell in circular formbut undergoes intracellular linearisation prior to chromosomalintegration. This is not necessarily an ideal situation since suchlinearisation may occur in a random fashion and potentially cleave theconstruct in a crucial region thereby rendering it ineffective.

The nucleic acid molecules which are utilised in the method of thepresent invention are derivable from any human or non-human source.Non-human sources contemplated by the present invention includeprimates, livestock animals (e.g., sheep, pigs, cows, goats, horses,donkeys), laboratory test animal (e.g., mice, hamsters, rabbits, rats,guinea pigs), domestic companion animal (e.g., dogs, cats), birds (e.g.,chicken, geese, ducks and other poultry birds, game birds, emus,ostriches) captive wild or tamed animals (e.g., oxes, kangaroos,dingoes), reptiles, fish, insects, prokaryotic organisms or syntheticnucleic acids.

It should be understood that the receptor-encoding constructs of thepresent invention may comprise nucleic acid material from more than onesource. For example, whereas the construct may originate from aparticular microorganism, in modifying that construct to introduce thefeatures defined herein, nucleic acid material from other microorganismsources may be introduced. These sources may include, for example, viralor bacterial DNA (eg. IRES DNA), mammalian DNA (e.g., the DNA encoding aCAR) or synthetic DNA (e.g., to introduce specific restrictionendonuclease sites). Still further, the cell type in which it isproposed to express the subject construct may be different again in thatit does not correspond to the same organism as all or part of thenucleic acid material of the construct. For example, a constructconsisting of essentially bacterial and viral derived DNA maynevertheless be expressed in the mammalian stem cells contemplatedherein.

Without limiting the present invention in any way, the present inventionpreferably uses a DNA construct comprising sequences of a CAR, whereinthe sequence comprises the nucleic acid sequence of an antigen bindingmoiety operably linked to the nucleic acid sequence of an intracellulardomain. For example, an intracellular domain that can be used in thesubject CAR includes but is not limited to the intracellular domain ofCD3-zeta. In another embodiment, the intracellular domain of a CARincludes the intracellular domain of CD3-zeta in operable linkage to theintracellular domain of CD28; and in a further embodiment, theintracellular domain of a CAR includes the intracellular domains ofCD3-zeta, CD28 and OX40, in operable linkage with each other.

Vectors derived from retroviruses such as the lentivirus are one exampleof vectors suitable to achieve long-term gene transfer since they allowlong-term, stable integration of a transgene and its propagation indaughter cells. Other suitable viruses include Sendai virus and Vacciniavirus. The vector should be suitable for replication and integrationinto eukaryotes. Typical cloning vectors contain transcription andtranslation terminators, initiation sequences, and promoters useful forregulation of the expression of the desired nucleic acid sequence. Viralvector technology is well known in the art and is described, forexample, in Sambrook et al (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in other virologyand molecular biology manuals. Viruses, which are useful as vectorsinclude, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses and lentiviruses. In general, asuitable vector contains an origin of replication functional in at leastone organism, a promoter sequence, convenient restriction endonucleasesites, and one or more selectable markers, (eg. WO 01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered to thesubject stem cells. A number of retroviral systems are known in the art.

Additional promoter elements, eg. enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,the individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1a(EF-1a). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theconstruct should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated to be used. The use of aninducible promoter provides a molecular switch capable of turning onexpression of the CAR polynucleotide sequence to which it is operativelylinked when such expression is desired, or turning off the expressionwhen expression is not desired. Examples of inducible promoters include,but are not limited to a metallothionine promoter, a glucocorticoidpromoter, a progesterone promoter, and a tetracycline promoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like. An epitope tag can also be included in theextracellular domain of a CAR molecule, such as the commonly used shortpolypeptide c-myc or FLAG, preferably placed within the hinge region, toidentify CAR expression by epitope specific targeting agents such asantibodies used in combination for example with flow cytometry.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al, 2000 FEBS Letters 479: 79-82). Suitable expression systemsare well known and may be prepared using known techniques or obtainedcommercially. In general, the construct with the minimal 5′ flankingregion showing the highest level of expression of reporter gene isidentified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription. It will be appreciated by those skilledin the art that a reporter such as eGFP (enhanced green fluorescentprotein) can be incorporated as a C-terminal polypeptide extension to aCAR, separated by a self-cleaving peptide such as P2A, which willrelease the reporter such as eGFP intracellularly.

Methods of introducing and expressing genes in a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell by physical, chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).A preferred method for the introduction of a polynucleotide into a hostcell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, eg., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle is a liposome(eg., an artificial membrane vesicle).

In the case where a non-viral delivery system is sought to be utilized,an exemplary delivery vehicle is a liposome. The use of lipidformulations is contemplated for the introduction of the nucleic acidsinto a host cell. In another aspect, the nucleic acid may be associatedwith a lipid. The nucleic acid associated with a lipid may beencapsulated in the aqueous interior of a liposome, interspersed withinthe lipid bilayer of a liposome, attached to a liposome via a linkingmolecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al(1991)). However, compositions that have different structures insolution than the normal vesicular structure are also encompassed. Forexample, the lipids may assume a micellar structure or merely exist asnonuniform aggregates of lipid molecules. Also contemplated arelipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, Southern and Northern blotting, RT-PCR andPCR or by detecting the presence or absence of a particular peptide,eg., by immunological means (ELISAs and Western blots).

The TCR and CAR, and antigen-binding receptors in some embodiments, ofthe present cells are each directed to an antigenic determinant.Reference to “antigenic determinant” should be understood as a referenceto any proteinaceous or non-proteinaceous molecule expressed by a cellwhich is sought to be targeted by the receptor-expressing T cells of thepresent invention. It would be appreciated that these are moleculeswhich may be “self” molecules in that they are normally expressed in thebody of a patient (such as would be expected on some tumour cells or anautoreactive cells) or they may be non-self molecules such as would beexpected where a cell is infected with a microorganism (eg. viralproteins). It should also be understood that the subject antigen is notlimited to antigens (whether self or not) which are naturally able toelicit a T or B cell immune response. Rather, in the context of thepresent invention, reference to “antigen” or “antigenic determinant” isa reference to any proteinaceous or non-proteinaceous molecule which issought to be targeted. As detailed hereinbefore, the target molecule maybe one to which the immune system is naturally tolerant, such as atumour antigen or auto-reactive immune cell antigen. However, it may bedesirable (even in light of potential collateral damage) to neverthelesstarget this antigen, for example to minimize the potentially even moresevere side effects which might be observed with a highly non-specificand systemic treatment, such as chemotherapy or immunosuppression, or toreduce the duration of treatment via a highly targeted treatment and/orto maximise the prospect of killing all unwanted cells. Preferably, saidmolecule is expressed on the cell surface.

It would be understood by the skilled person that in the context of TCRbinding, the subject antigenic determinant will take the form of apeptide derived from an antigen, which peptide is expressed in thecontext of either MHC I or MHC II. In the context of the CAR, since thedesign of this receptor is based on the use of an immunoglobulinvariable region binding domain, the receptor will recognise an epitopepresent on the native form of the antigen. The subject epitope may beeither linear or conformational. It should be understood that thesubject antigenic determinant may be any molecule expressed by the cellwhich is sought to be targeted. That is, the molecule which is targetedmay be exclusively expressed by the target cell or it may also beexpressed by non-target cells too. Preferably, the subject antigenicdeterminant is a non-self antigenic determinant or an antigenicdeterminant which is otherwise expressed exclusively, or at asignificantly higher level than by normal cells, by the cells which aresought to be targeted. However, as discussed hereinbefore, depending onthe disease condition to be treated, it may not always be possible toidentify and target a non-self antigenic determinant.

Reference herein to TCR/CAR receptors which are directed to a “first”antigenic determinant and to a “second” antigenic determinant should beunderstood as a reference to the fact that the subject receptors aredirected to two different epitopic regions. In this regard, however, itshould be understood that the receptors may be directed to epitopes ontwo entirely different cell surface molecules or the receptors may bedirected to two different regions/epitopes of the same cell surfacemolecule. In embodiments where reference is made to a TCR together withmultiple CARs, or where reference is made to a TCR with one or more CARsand one or more antigen-binding receptors, it should be understood thateach receptor is directed to an antigenic determinant, and the antigenicdeterminants are preferably different from one another, i.e., theantigenic determinants corresponding to different epitopic regions ofthe same or different molecules.

Accordingly in one embodiment there is provided a genetically modifiedmammalian stem cell, or T cell differentiated therefrom, which cellexpresses at least one homozygous HLA haplotype, is capable ofdifferentiating to a T cell expressing a TCR directed to a firstantigenic determinant, and comprises at least one (i.e., one or more)nucleic acid molecule encoding a chimeric antigen receptor, wherein saidreceptor comprises an antigen recognition moiety directed to a secondantigenic determinant, which antigen recognition moiety is operablylinked to a T cell activation moiety, and optionally further comprises anucleic acid encoding an antigen-binding receptor directed to a thirdantigenic determinant, and wherein said antigenic determinants areselected from tumour antigens, microorganism antigens or autoreactiveimmune cell antigens.

In one embodiment, said stem cell is an iPSC. In another embodiment, thestem cell is an HSC.

In still another embodiment, said stem cell is capable ofdifferentiating to a CD4⁺ T cell or a CD8⁺ T cell.

In still another embodiment, said TCR is an αβ TCR.

In yet still another embodiment, said stem cell such as iPSC derivedfrom a T cell or thymocyte, preferably a CD8⁺ T cell or thymocyte.

As would be appreciated by the skilled person, the identification ofantigens which are exclusive to tumours is a significant area ofresearch, but in respect of which there has been limited progress. Sincetumour cells are usually self cells, (as opposed to, for example,tumours arising from transplant tissues), it is the case that theantigens which they express are not only self antigens, but are likelyto also be expressed by the non-neoplastic cells of the tissue fromwhich the tumour is derived. This is clearly a less than ideal situationdue to the side-effects (in terms of destruction of non-neoplastictissue) which can arise when an anti-neoplastic treatment regime istargeted to such an antigen, but is unavoidable. Nevertheless, someprogress has been made in terms of identifying target tumour antigenswhich, even if not expressed exclusively by tumour cells, are expressedat lower levels or otherwise less frequently on non-neoplastic cells.

The selection of the antigen binding moiety of the invention will dependon the particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, MAGE, LMP-2, CD19, CD20, WT1,MART-1 glioma-associated antigen, carcinoembryonic antigen (CEA),β-human chorionic gonadotropin, tumour associated glycoprotein 72 (TAG72), alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrinB2, CD22, insulingrowth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin. CD47(“don't eat me” receptor) is also a tumour target because it is oftenhighly expressed in cancer cells, as compared to normal cells, andprevents these cancer cells from being attacked by cells of the immunesystem including, and in particular, scavenger macrophages.

In one embodiment, the tumor antigen comprises one or more epitopesassociated with a malignant tumor. Malignant tumors express a number ofproteins that can serve as target antigens for an immune attack. Thesemolecules include but are not limited to tissue-specific antigens suchas MART-1, WT-1, tyrosinase and GP 100 in melanoma and prostatic acidphosphatase (PAP) and prostate-specific antigen (PSA) in prostatecancer. Other target molecules belong to the group oftransformation-related molecules such as the oncogene HER-2/Neu/ErbB-2.Yet another group of target antigens are onco-fetal antigens such ascarcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specificidiotype immunoglobulin constitutes a truly tumor-specificimmunoglobulin antigen that is unique to the individual tumor. B-celldifferentiation antigens such as CD 19, CD20 and CD37 are othercandidates for target antigens in B-cell lymphoma.

Non-limiting examples of antigens include the following: Differentiationantigens such as MART-1/MelanA (MART-I), gp1OO (Pmel 17), tyrosinase,TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1,MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens suchas CEA; overexpressed oncogenes and mutated tumor-suppressor genes suchas p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomaltranslocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; andviral antigens, such as the Epstein Barr virus antigens EBVA and thehuman papillomavirus (HPV) antigens E6 and E7. Other large,protein-based antigens include CD47, TSP-180, MAGE-4, MAGE-5, MAGE-6,RAGE, NY-ESO, pl 85erbB2, p180erbB-3, cMet, nm-23H1, PSA, TAG-72, CA19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA 15-3\CA 27. 29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1,CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV 18,NB/70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 bindingprotein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

The cells of the present invention are designed to be directed tomultiple, i.e., two or more, antigenic determinants. As detailed herein,the multiple antigenic determinants may be, or include, in someembodiments, multiple epitopes of one molecule, or, in otherembodiments, epitopes of multiple entirely distinct molecules. Theselection of which multiple antigenic determinants should be targetedand, further, whether they should be targeted by the TCR or the CAR iswell within the skill of the person in the art. In one embodiment, thecells of the present invention are designed to clear tumour cells andsaid TCR/CAR are directed to tumour antigens, in particular TAG 72, MAGEand WT1. In another embodiment, said cells are designed to clearautoreactive immune cells and said TCR/CAR are directed to idiotypic Tcell or B cell receptors.

Accordingly, in one embodiment there is provided a genetically modifiedmammalian stem cell, or T cell differentiated therefrom, which cell iscapable of differentiating to a T cell expressing a TCR directed to afirst tumour antigenic determinant, and comprises one or more nucleicacid molecules encoding one or more chimeric antigen receptors, whereineach chimeric antigen receptor comprises an antigen recognition moietydirected to a tumour antigenic determinant, which antigen recognitionmoiety operably linked to a T cell activation moiety and wherein saidantigenic determinants are selected from TAG 72, CD47, CD19, WT-1, MAGEand EBVLMP2.

Preferably, said genetically modified cell is directed to TAG72 andWT-1. Still more preferably, said CAR is directed to TAG72 and CD47, andsaid TCR is directed to WT-1.

In one embodiment, said stem cell is an iPSC. In another embodiment, thestem cell is an HSC.

In still another embodiment, said stem cell is capable ofdifferentiating to a CD4⁺ T cell or a CD8⁺ T cell.

In still another embodiment, said TCR is an αβ TCR.

In yet still another embodiment, said stem cell (such as iPSC) isderived from a T cell or thymocyte, preferably a CD8⁺ T cell orthymocyte.

To the extent that the cells of the present invention, in oneembodiment, are directed to treating neoplasias, a wide range of CARshave been developed to target known tumour antigens. A non-limitingsummary exemplifying some of these CARs, together with the structure ofthe receptor, is provided in Table 5, below:

TABLE 5 Target antigen Associated malignancy Receptor type α-Folatereceptor Ovarian cancer ScFv-FcεRIγCAIX CAIX Renal cell carcinomaScFv-FcεRIγ CAIX Renal cell carcinoma ScFv-FcεRIγ CD19 B-cellmalignancies ScFv-CD3ζ (EBV) CD19 B-cell malignancies, CLL ScFv-CD3ζCD19 B-ALL ScFv-CD28-CD3ζ CD19 ALL CD3ζ(EBV) CD19 ALL post-HSCTScFv-CD28-CD3ζ CD19 Leukemia, lymphoma, CLL ScFv-CD28-CD3ζ vs. CD3ζ CD19B-cell malignancies ScFv-CD28-CD3ζ CD19 B-cell malignancies post-HSCTScFv-CD28-CD3ζ CD19 Refractory Follicular Lymphoma ScFv-CD3ζ CD19 B-NHLScFv-CD3ζ CD19 B-lineage lymphoid malignancies post-UCBT ScFv-CD28-CD3ζCD19 CLL, B-NHL ScFv-CD28-CD3ζ CD19 B-cell malignancies, CLL, B-NHLScFv-CD28-CD3ζ CD19 ALL, lymphoma ScFv-41BB-CD3ζ vs CD3ζ CD19 ALLScFv-41BB-CD3ζ CD19 B-cell malignancies ScFv-CD3ζ (Influenza MP-1) CD19B-cell malignancies ScFv-CD3ζ (VZV) CD20 Lymphomas ScFv-CD28-CD3ζ CD20B-cell malignancies ScFv-CD4-CD3ζ CD20 B-cell lymphomas ScFv-CD3ζ CD20Mantle cell lymphoma ScFv-CD3ζ CD20 Mantle cell lymphoma, indolent B-NHLCD3 ζ/CD137/CD28 CD20 indolent B cell lymphomas ScFv-CD28-CD3ζ CD20Indolent B cell lymphomas ScFv-CD28-41BB-CD3ζ CD22 B-cell malignanciesScFV-CD4-CD3ζ CD30 Lymphomas ScFv-FcεRIγ CD30 Hodgkin lymphoma ScFv-CD3ζ(EBV) CD33 AML ScFv-CD28-CD3ζ CD33 AML ScFv-41BB-CD3ζ CD44v7/8 Cervicalcarcinoma ScFv-CD8-CD3ζ CEA Breast cancer ScFv-CD28-CD3ζ CEA Colorectalcancer ScFv-CD3ζ CEA Colorectal cancer ScFv-FceRIγ CEA Colorectal cancerScFv-CD3ζ CEA Colorectal cancer ScFv-CD28-CD3ζ CEA Colorectal cancerScFv-CD28-CD3ζ EGP-2 Multiple malignancies scFv-CD3ζ EGP-2 Multiplemalignancies scFv-FcεRIγ EGP-40 Colorectal cancer scFv-FcεRIγ erb-B2Colorectal cancer CD28/4-1BB-CD3ζ erb-B2 Breast and othersScFv-CD28-CD3ζ erb-B2 Breast and others ScFv-CD28-CD3ζ (Influenza)erb-B2 Breast and others ScFv-CD28mut-CD3ζ erb-B2 Prostate cancerScFv-FcεRIγ erb-B 2, 3, 4 Breast and others Heregulin-CD3ζ erb-B 2, 3, 4Breast and others ScFv-CD3ζ FBP Ovarian cancer ScFv-FcεRIγ FBP Ovariancancer ScFv-FcεRIγ (alloantigen) Fetal acetylcholine receptorRhabdomyosarcoma ScFv-CD3ζ GD2 Neuroblastoma ScFv-CD28 GD2 NeuroblastomaScFv-CD3ζ GD2 Neuroblastoma ScFv-CD3ζ GD2 NeuroblastomaScFv-CD28-OX40-CD3ζ GD2 Neuroblastoma ScFv-CD3ζ (VZV) GD3 MelanomaScFv-CD3ζ GD3 Melanoma ScFv-CD3ζ Her2/neu Medulloblastoma ScFv-CD3ζHer2/neu Lung malignancy ScFv-CD28-CD3ζ Her2/neu Advanced osteosarcomaScFv-CD28-CD3ζ Her2/neu Glioblastoma ScFv-CD28-CD3ζ IL-13R-a2 GliomaIL-13-CD28-4-1BB-CD3ζ IL-13R-a2 Glioblastoma IL-13-CD3ζ IL-13R-a2Medulloblastoma IL-13-CD3ζ KDR Tumor neovasculature ScFv-FcεRIγ k-lightchain B-cell malignancies ScFv-CD3ζ k-light chain (B-NHL, CLL)ScFv-CD28-CD3ζ vs CD3ζ LeY Carcinomas ScFv-FcεRIγ LeY Epithelial derivedtumors ScFv-CD28-CD3ζ L1 cell adhesion molecule Neuroblastoma ScFv-CD3ζMAGE-A1 Melanoma ScFV-CD4-FcεRIγ MAGE-A1 Melanoma ScFV-CD28-FcεRIγMesothelin Various tumors ScFv-CD28-CD3ζ Mesothelin Various tumorsScFv-41BB-CD3ζ Mesothelin Various tumors ScFv-CD28-41BB-CD3ζ Murine CMVinfected cells Murine CMV Ly49H-CD3ζ MUC1 Breast, OvaryScFV-CD28-OX40-CD3ζ NKG2D ligands Various tumors NKG2D-CD3ζ Oncofetalantigen (h5T4) Various tumors ScFV-CD3ζ (vaccination) PSCA Prostatecarcinoma ScFv-b2c-CD3ζ PSMA Prostate/tumor vasculature ScFv-CD3ζ PSMAProstate/tumor vasculature ScFv-CD28-CD3ζ PSMA Prostate/tumorvasculature ScFv-CD3ζ TAA targeted by mAb IgE Various tumorsFceRI-CD28-CD3ζ (+a-TAA IgE mAb) TAG-72 Adenocarcinomas scFv-CD3ζVEGF-R2 Tumor neovasculature scFv-CD3ζ

In some embodiments, a CAR comprises an antigen recognition domain whichis comprised of an scFv directed to CD19 or TAG-72, and a hinge (Stalk)region and a transmembrane region both of which are derived from CD28 orCD8, and a cytoplasmic endodomain which is also derived from CD28 or CD8and comprises a T cell activation moiety. The CAR can include a reporterprotein (such as EGFP) as a C-terminal polypeptide extension, joinedtogether by a P2A self-cleaving polypeptide to release EGFP aftertranslation. See, e.g., FIGS. 11 and 14.

In a related aspect, it has been further determined that the cells ofthe present invention are rendered particularly effective if they areengineered to express a non-signalling antigen-binding receptor, forexample, a CD47 binding molecule which is unable to effect signaltransduction. The expression of a CD47 binding molecule on the cellsurface anchors the cell of the present invention to the neoplastic cellto which it is directed, thereby facilitating improved interaction ofthe TCR and the CAR with their respective ligands. In terms of thetreatment of solid tumours, in particular, the increased stability andbinding affinity of the interaction of the subject cell enables improvedfunctional outcomes, in terms of neoplastic cell killing, relative to acell which does not express the subject CD47 binding molecule.

Accordingly, in a related aspect of the present invention there isprovided a genetically modified mammalian stem cell, or T celldifferentiated therefrom, which cell is capable of differentiating to aT cell expressing a TCR directed to a first antigenic determinant, andcomprises (i) a nucleic acid molecule encoding a chimeric antigenreceptor, wherein said receptor comprises an antigen recognition moietydirected to a second antigenic determinant, which antigen recognitionmoiety is operably linked to a T cell activation moiety and (ii) anucleic acid molecule encoding a non-signalling antigen-bindingreceptor, such as a non-signalling CD47 binding receptor. In someembodiments, the genetically modified mammalian stem cell expresses atleast one homozygous HLA haplotype.

Without limiting the present invention to any one theory or mode ofaction, CD47 (also known as integrin associated protein) is atransmembrane protein that in humans is encoded by the CD47 gene. CD47belongs to the immunoglobulin superfamily. CD47 is involved in a rangeof cellular processes, including apoptosis, proliferation, adhesion, andmigration. Furthermore, it plays a key role in immune and angiogenicresponses. CD47 is ubiquitously expressed in human cells and has beenfound to be overexpressed in many different tumor cells.

CD47 is a 50 kDa membrane receptor that comprises an extracellularN-terminal IgV domain, five transmembrane domains, and a shortC-terminal intracellular tail. There are four alternatively splicedisoforms of CD47 that differ only in the length of their cytoplasmictail. Form 2 is the most widely expressed form that is found in allcirculating and immune cells. The second most abundant isoform is form4, which is predominantly expressed in the brain and in the peripheralnervous system. Only keratinocytes express significant amounts ofform 1. These isoforms are highly conserved between mouse and man,suggesting an important role for the cytoplasmic domains in CD47function.

CD47 is a receptor for thrombospondin-1 (TSP-1), a secreted glycoproteinthat plays a role in vascular development and angiogenesis. Binding ofTSP-1 to CD47 influences several fundamental cellular functionsincluding cell migration and adhesion, cell proliferation or apoptosis,and plays a role in the regulation of angiogenesis and inflammation.CD47 also interacts with signal-regulatory protein alpha (SIRPα), aninhibitory transmembrane receptor present on myeloid cells. TheCD47/SIRPα interaction leads to bidirectional signalling, resulting indifferent cell-to-cell responses including inhibition of phagocytosis(facilitating cancer cell escape), stimulation of cell-cell fusion, andT-cell activation. Still further, CD47 interacts with several membraneintegrins, most commonly integrin avb3. These interactions result inCD47/integrin complexes that affect a range of cell functions includingadhesion, spreading and migration.

However, although CD47 is ubiquitously expressed, it has been determinedthat the increased level of expression of CD47 on neoplastic cells issufficient to facilitate improved responsiveness to, and clearing of,said neoplastic cells by molecules targeting CD47, prior to anysubstantive adverse impact on non-neoplastic cells.

Reference to a “binding receptor” directed to CD47 should be understoodas a reference to any receptor which interacts with CD47. This may takethe form of a CD47-binding receptor such as a surface-displayed antibodyfragment, for example, and preferably lacking a signalling function.

Accordingly to this embodiment, there is provided a genetically modifiedmammalian stem cell, or T cell differentiated therefrom, which cell iscapable of differentiating to a T cell expressing a TCR directed to afirst antigenic determinant, and comprises (i) a nucleic acid moleculeencoding a chimeric antigen receptor, wherein said receptor comprises anantigen recognition moiety directed to a second antigenic determinant,which antigen recognition moiety is operably linked to a T cellactivation moiety and (ii) a nucleic acid molecule encoding anon-signalling antigen-binding receptor, wherein said receptor comprisesan antigen recognition moiety directed to CD47. In some embodiments, thegenetically modified mammalian stem cell expresses at least onehomozygous HLA haplotype.

As detailed hereinbefore, the subject CD47 binding receptor is anon-signalling receptor. By “non-signalling” is meant that subsequentlyto binding of the subject receptor to CD47 on a target cell, there is nosignal transmitted which would effect a change to the functionality ofthe cell of the present invention. Rather, the purpose of the CD47binding receptor is to provide improved anchoring of the subject cell toa target cell, thereby improving the effectiveness of binding of the TCRand the CAR which is directed to the target antigen moiety, such as atumour antigen moiety.

For example, in one design of a non-signalling antigen-binding receptor,the extracellular domain of the receptor comprises an antigenrecognition moiety with binding specificity to CD47, a hinge (stalk)domain, a transmembrane domain, and an intracellular domain whichcompletely lacks a cytoplasmic signalling function. Such non-signallingCD47 binding receptor can be used simply for attachment, not forsignalling, so it can drive the docking of T-cells to cancer cells viaCD47 binding and without the unwanted activation and kill if engaging tonormal CD47-expressing cells.

In some embodiments, the antigen recognition moiety of a non-signallingCD47-binding receptor includes antibody-like domains such as scFv, Fv,Fab etc and any CD47 targeted V-domain, including single human andmammalian V-domains and their equivalent (VhH or vNAR) domains, or mayinclude “alternative protein-based targeting scaffolds” that are wellknown in the field including, but not restricted to, darpins,anticalins, knottins, ImmE7s, affibodies, Fn3 fibronectin domains etc.The antigen recognition moiety may also include one or more of theV-like domains of SIRPa (the natural ligand of CD47). In one embodiment,the antigen recognition moiety may include one natural V-like domain ofSIRPα. In another embodiment, the antigen recognition moiety may includeall three of the natural V-like domain of SIRPα. In other embodiments, amolecule suitable for use to provide an antigen recognition moiety in anon-signalling CD47 binding receptor is Hu5F9-G4 scFv molecule(described in U.S. patent application Ser. No. 14/656,431). Hu5F9 hasbeen designed with 3 different versions of VH (1,2,3) and 3 differentversions of VL (11, 12, 13), shown in FIGS. 12A, 12B of U.S. patentapplication Ser. No. 14/656,431, published as US 20150183874 A1. Liu etal (PLOS One (2015) September 21;10(9):e0137345) describes Hu5F9-G4where the selected V-domains were heavy VH-2 comprising 4 unique residuechanges in the framework (that differentiate VH-2 from VH-1,3) and lightVL-12 comprising 2 unique residue changes in the framework (thatdifferentiate VL-12 from VL-11,13).

In some embodiments, the hinge region of a non-signalling CD47-bindingreceptor can be the natural SIRPα hinge sequence, or the CD8 or CD28hinges as typically used in CARs, or alternative hinges well known inthe field such as CD4 domains or Mucin peptide hinges. The hinge regioncan be designed to include one or more Cysteine (Cys) residues in orderto allow for dimerization of the receptors. CD28 is a natural dimericstructure linked via a single Cys in the stalk region. Thus, where thestalk region of CD28 is used as the hinge of non-signalling CD47-bindingreceptor, introduction of an additional Cys may not be necessary, butmay provide additional stabilization for dimers.

It will be understood by those skilled in the art that the introductionof nucleic acids encoding a CAR and a non-signalling antigen-bindingreceptor (such as a non-signalling CD47-binding receptor) into a cell(e.g., a T cell or an iPSC) can be achieved using two separatetransfection vectors, or a single bicistronic vector, or a single geneencoding an internal cleavage signal to separate the CAR from theantigen-binding receptor. In one embodiment the internal cleavage signalis P2A, a peptide sequence that directs self-cleavage to separate CARfrom the antigen-binding receptor. In a specific embodiment, anon-signalling CD47 binding receptor is expressed as a C-terminalextension of a CAR and separated by a P2A self-cleaving peptide toseparate the CAR and the CD47-binding receptor after translation.

Means for modifying the stem cell of the present invention, such that italso expresses a non-signalling CD47 binding molecule, have beendescribed in significant detail hereinbefore in terms of effecting theexpression of a chimeric antigen receptor directed to a tumour antigenmoiety. The transfection and other methods of achieving receptorexpression which are described herein would be understood by the skilledperson to be equally applicable in the context of the subject CD47binding molecule.

In one embodiment, said stem cell is an iPSC. In another embodiment,said stem cell is an HSC.

In another embodiment, said stem cell is capable of differentiating to aCD4⁺ T cell or a CD8⁺ T cell.

In still another embodiment, said TCR is an αβ TCR.

In yet still another embodiment, said stem cell such as iPSC is derivedfrom a T cell or thymocyte, preferably a CD8⁺ T cell or thymocyte, andin some embodiments, a CD8⁺ T cell or thymocyte with an endogenous TCRdirected to a tumour antigen.

In still yet another embodiment, said stem cell is directed to TAG 72and WT 1. Still more preferably, said CAR is directed to TAG 72 and saidTCR is directed to WT 1.

In a further aspect there is provided a method of making a geneticallymodified mammalian stem cell. The various means for making a geneticallymodified mammalian stem cell, particularly an iPSC have been describedhereinabove.

In a further aspect there is provided a T cell that expresses a TCRdirected to a first antigenic determinant, and a chimeric antigenreceptor, wherein said receptor comprises an antigen recognition moietydirected to a second antigenic determinant, which antigen recognitionmoiety is operably linked to a T cell activation moiety. In someembodiments, the T cell expresses at least one homozygous HLA haplotype.

In one embodiment, the T cell expresses multiple chimeric antigenreceptors, wherein each chimeric antigen receptor comprises an antigenrecognition moiety directed to an antigenic determinant, which antigenrecognition moiety is operably linked to a T cell activation moiety.

In one embodiment, the multiple antigenic determinants which themultiple chimeric antigen receptors are directed to are each distinctfrom said first antigenic determinant to which the TCR expressed on thesubject T cell is directed. In another embodiment, the multipleantigenic determinants which the multiple chimeric antigen receptors aredirected to, are distinct one from another, and are also distinct fromsaid first antigenic determinant to which the TCR expressed on thesubject T cell is directed.

In one embodiment, the multiple CARs are encoded by one contiguousnucleic acid fragment. For example, the multiple CARs are encoded bymultiple nucleic acids placed in one vector, which is transfected into acell to ultimately generate the subject T cell. In a specificembodiment, the multiple CAR encoding nucleic acids can be linked toeach other within one expression unit and reading frame (for example, byutilizing a self-cleaving peptide such as P2A), such that one singlepolypeptide comprising multiple CAR polypeptide sequences is initiallyproduced and subsequently processed to provide multiple CARs. In anotherembodiment, the multiple CAR-encoding nucleic acids are placed inseparate vectors, which are used in transfection to generate the subjectT cell.

In another embodiment, the T cell, which expresses one or more CARs,further expresses at least one (i.e., one or more) antigen-bindingreceptor which comprises an antigen recognition moiety directed to athird antigenic determinant.

In one embodiment, the antigen-binding receptor is a non-signallingantigen-binding receptor; namely, the receptor is anchored to the cellsurface of the subject T cell and binds to the third antigenicdeterminant, but does not transduce signal into the cytoplasmic part ofthe T cell that would affect the function of the T cell (hence alsoreferred to as a non-T cell signalling antigen-binding receptor). In oneembodiment, the antigen-binding receptor comprises an antigenrecognition moiety directed to a third antigenic determinant, operablylinked to a transmembrane domain, but lacks a T cell activation moiety.

In a specific embodiment, the antigen-binding receptor is anon-signalling antigen-binding receptor directed to CD47. For example,the antigen-binding receptor is a non-signalling CD47-binding molecule.

In some embodiments, the T cell provided herein is CD4+. In otherembodiments, the T cell is CD8+.

In some embodiments, the T cell provided herein expresses an αβ TCR. Inother embodiments, the T cell provided herein expresses a γδ TCR.

In some embodiments, the multiple antigenic determinants to which thesubject T cell is directed, i.e., the first antigenic determinant towhich the TCR is directed, the antigenic determinant(s) to which thechimeric antigen receptor(s) is(are) directed, and the antigenicdeterminant(s) to which the antigen-binding receptor(s) is(are) directedif such antigen-binding receptor(s) is(are) present, can be selectedfrom tumour antigens, microorganism antigens, or autoreactive immunecell antigens. In certain embodiments, the antigenic determinants areselected from tumour antigens. In specific embodiments, the antigenicdeterminant to which the TCR is directed, is selected from TCRrecognized peptides such as WT-1 or EbvLMP2. In other specificembodiments, the antigenic determinants to which a chimeric antigenreceptor and an antigen-binding receptor are directed, can be selectedfrom for example, TAG-72, CD19, MAGE, or CD47.

In some embodiments, the subject T cell, which expresses a TCR directedto a first antigenic determinant, and expresses a chimeric antigenreceptor which comprises an antigen recognition moiety directed to asecond antigenic determinant, operably linked to a T cell activationmoiety, is derived from an iPSC or an HSC.

In one embodiment, the iPSC or HSC from which the subject T cell isderived, is a genetically modified iPSC or HSC which is capable ofdifferentiating into a T cell which expresses a TCR directed to saidfirst antigenic determinant, and comprises one or more nucleic acid(s)encoding one or more chimeric antigen receptor, and optionally comprisesone or more nucleic acid encoding an antigen-binding receptor(s). Inanother embodiment, the iPSC or HSC from which the subject T cell isderived, is capable of differentiating into a T cell which expresses aTCR directed to said first antigenic determinant; and one or morenucleic acid(s) encoding one or more chimeric antigen receptor, andoptionally one or more nucleic acid encoding an antigen-bindingreceptor(s), are introduced after the iPSC or HSC has differentiatedinto a T cell. In some embodiments, the iPSC or HSC from which thesubject T cell is derived, expresses at least one HLA haplotype, and theT cell derived from such iPSC or HSC also expresses said at least oneHLA haplotype.

In one embodiment, the iPSC from which the subject T cell is derived, isitself derived from a T cell or thymocyte. In one embodiment, the iPSCis derived from a CD8+ T cell or thymocyte. In one embodiment, the iPSCis derived from a T cell or thymocyte, which expresses a TCR directed tothe first antigenic determinant, i.e., the same antigenic determinant towhich the TCR of the subject T cell derived from the iPSC is directed.

The value of the cells of the present invention is predicated ondirecting the differentiation of the subject stem cell to a CD4⁺ or CD8⁺T cell. In this regard, reference to “directing” the differentiation ofa stem cell to a T cell should be understood to mean that a cell culturesystem is applied which induces commitment of a stem cell to the T celllineage and differentiation along that lineage to a mature T cell. Meansfor effecting the directed differentiation of a stem cell along the Tcell lineage are well known to those of skill in the art. For example,and as exemplified herein, the introduction of Notch-dependentsignalling into the culture system is known to effect the directeddifferentiation of stem cells along the T cell lineage. Still further,if this signalling is provided to stem cells in the context of theirco-culture over the OP-9 feeder cell layer, particularly efficientdifferentiation is achieved. Examples of Notch ligands which aresuitable for use include, but are not limited to, Delta-like 1, andDelta-4. In this regard, OP-9 cells have been engineered to expressDelta-like 1 (OP9-DL1), thereby providing a highly convenient means ofgenerating T cells from stem cells. In another example, and asexemplified herein, the subject stem cells are first cultured infeeder-free conditions to generate mesoderm, followed by co-culture onthe OP9-DL1 cell line. A particularly preferred method of achieving thedirected differentiation to CD8⁺ T cells is exemplified herein.

In another aspect there is provided a method for making a T cell thatexpresses a TCR directed to a first antigenic determinant, and expressesone or more CARs, and optionally one or more antigen-binding receptors.In some embodiments, the T cell also expresses at least one homozygousHLA haplotype.

In one embodiment, the method comprises obtaining a genetically modifiedstem cell (such as a genetically modified iPSC or HSC) which is capableof differentiating into a T cell which expresses a TCR directed to afirst antigenic determinant, and comprises one or more nucleic acid(s)encoding one or more chimeric antigen receptor each directed to anantigenic determinant (preferably distinct from the first antigenicdeterminant), and optionally further comprises one or more nucleic acidencoding one or more antigen-binding receptor(s) each directed to anantigenic determinant (preferably distinct from the first antigenicdeterminant); and differentiating such genetically modified stem cellinto a T cell. In some embodiments, the genetically modified stem cellalso expresses at least one homozygous HLA haplotype.

In another embodiment, the method comprises obtaining a stem cell (suchas an iPSC or HSC) which is capable of differentiating into a T cellwhich expresses a TCR directed to a first antigenic determinant;differentiating the stem cell into a T cell; introducing into the T cellone or more nucleic acid(s) encoding one or more chimeric antigenreceptor, each directed to an antigenic determinant (preferably distinctfrom the first antigenic determinant), and optionally also one or morenucleic acid encoding one or more antigen-binding receptor(s) eachdirected to an antigenic determinant (preferably distinct from the firstantigenic determinant). In some embodiments, the genetically modifiedstem cell (such as an iPSC or HSC) also expresses at least onehomozygous HLA haplotype.

Irrespective of whether a CAR-encoding nucleic acid is introduced into astem cell before differentiation into a T cell, or introduced into a Tcell after differentiation from a stem cell, the stem cell (such as aniPSC) can be itself derived from a T cell or thymocyte. Such T cell andthymocyte can have a TCR specific for a nominal antigen, e.g., a tumourantigen. In one embodiment, the stem cell is an iPSC. In one embodiment,the iPSC is derived from a CD8+ T cell or thymocyte. In anotherembodiment, the iPSC is derived from a T cell or thymocyte expressing aTCR directed to the same antigenic determinant to which the TCRexpressed on the T cell derived from the iPSC is directed.

Reference to “mammal” should be understood to include reference to amammal such as but not limited to human, primate, livestock animal(e.g., sheep, cow, horse, donkey, pig), companion animal (e.g., dog,coat), laboratory test animal (e.g., mouse, rabbit, rat, guinea pig,hamster), captive wild animal (e.g., fox, deer). Preferably the mammalis a human or primate. Most preferably the mammal is a human.

The development of the present invention has now facilitated thedevelopment of means for treating disease conditions characterised bythe presence of an unwanted cellular population such as a neoplasticpopulation of cells, virally infected cells, autoreactive immune cellsor infection with microorganisms such as antibiotic resistant bacteria.More specifically, the cells of the present invention provide a means ofclearing these cells in a more targeted fashion than current highlynon-specific methods such as chemotherapy to treat a neoplasticcondition, anti-inflammatory therapy to treat the symptoms of autoimmunedisease or immunosuppression to manage autoimmunity. In this regard,reference to a disease condition “characterised by the presence of anunwanted cellular population” should be understood as a reference to anycondition, a symptom or cause of which is the presence or functioning ofa population of cells which can be targeted by virtue of an expressedcell surface antigen and the elimination of some or all of which cellswould be beneficial to the patient. Treatment of the subject conditionis achieved by administering T cells differentiated from the stem cellsof the present invention, the dual TCR/CAR of which T cells are directedto two or more antigenic determinants expressed by the cells which aresought to be cleared.

It should be understood that the “cells” which are sought to be clearedby the T cells of the present invention may be any cell, whether self ornon-self. For example, to the extent that the T cells of the presentinvention are designed to treat a disease condition such as a neoplasia,viral infection or autoimmune disease, the target population of cellswhich are sought to be cleared are self cells. However, to the extentthat the condition which is sought to be treated is, for example,infection by a microorganism, such as antibiotic resistant bacteria or aparasite, the “cell” to be cleared is a foreign cell. In this regard,the cell may be in suspension (such as leukaemic cells which are presentin the circulation) or they may be part of a mass (such as a tumour ortissue). To the extent that the condition being treated is amicroorganism infection, the cells may correspond to a unicellularmicroorganism (such as many bacteria) or they may be part of amulticellular organism. The T cells of the present invention are usefulfor targeting any type of cell which presents in any type of formation.

Accordingly, another aspect of the present invention is directed to amethod of treating a condition characterised by the presence of anunwanted population of cells in a mammal, said method comprisingadministering to said mammal an effective number of stem cells or Tcells differentiated therefrom, as hereinbefore defined.

In one embodiment, said condition is a neoplastic condition, amicroorganism infection (such as HIV, STD or antibiotic resistantbacteria), or an autoimmune condition.

In another embodiment, said stem cell is an iPSC or an HSC.

In still another embodiment, said stem cell is capable ofdifferentiating to a CD4⁺ T cell or a CD8⁺ T cell.

In still another embodiment, said TCR is an αβ TCR.

In yet still another embodiment, said stem cell such as iPSC is derivedfrom a T cell or thymocyte.

In still another embodiment, the cell further comprises a nucleic acidmolecule encoding a non-signalling antigen-binding receptor, whereinsaid receptor comprises an antigen recognition moiety directed to CD47.

According to these embodiments, in one particular aspect there isprovided a method of treating a neoplastic condition, said methodcomprising administering to said mammal an effective number of stemcells, or T cells differentiated therefrom, as hereinbefore definedwherein said TCR is directed to a first tumour antigenic determinant andsaid CAR is directed to one or more additional tumour antigenicdeterminant(s).

In one embodiment, said first tumour antigenic determinant is WT 1.

In another embodiment, said second tumour antigenic determinant isTAG72.

In another embodiment, the cell further comprises a nucleic acidmolecule encoding a non-signalling antigen-binding receptor, whereinsaid receptor comprises an antigen recognition moiety directed to CD47.

In another embodiment the genetically modified stem cell also expressesat least one homozygous HLA haplotype.

Reference to a “neoplastic condition” should be understood as areference to a condition characterised by the presence or development ofencapsulated or unencapsulated growths or aggregates of neoplasticcells. Reference to a “neoplastic cell” should be understood as areference to a cell exhibiting abnormal growth. The term “growth” shouldbe understood in its broadest sense and includes reference toenlargement of neoplastic cell size as well as proliferation.

The phrase “abnormal growth” in this context is intended as a referenceto cell growth which, relative to normal cell growth, exhibits one ormore of an increase in individual cell size and nuclear/cytoplasmicratio, an increase in the rate of cell division, an increase in thenumber of cell divisions, a decrease in the length of the period of celldivision, an increase in the frequency of periods of cell division oruncontrolled proliferation and evasion of apoptosis. Without limitingthe present invention in any way, the common medical meaning of the term“neoplasia” refers to “new cell growth” that results as a loss ofresponsiveness to normal growth controls, eg. to neoplastic cell growth.Neoplasias include “tumours” which may be benign, pre-malignant ormalignant. The term “neoplasm” should be understood as a reference to alesion, tumour or other encapsulated or unencapsulated mass or otherform of growth or cellular aggregate which comprises neoplastic cells.

The term “neoplasm”, in the context of the present invention should beunderstood to include reference to all types of cancerous growths oroncogenic processes, metastatic tissues or malignantly transformedcells, tissues or organs irrespective of histopathologic type or stateof invasiveness.

The term “carcinoma” is recognised by those skilled in the art andrefers to malignancies of epithelial or endocrine tissues includingrespiratory system carcinomas, gastrointestinal system carcinomas,genitourinary system carcinomas, testicular carcinomas, breastcarcinomas, prostate carcinomas, endocrine system carcinomas andmelanomas. The term also includes carcinosarcomas, e.g. which includemalignant tumours composed of carcinomatous and sarcomatous tissues. An“adenocarcinoma” refers to a carcinoma derived from glandular tissue orin which the tumour cells form recognisable glandular structures.

The neoplastic cells comprising the neoplasm may be any cell type,derived from any tissue, such as an epithelial or non-epithelial cell.Reference to the terms “malignant neoplasm” and “cancer” and “carcinoma”herein should be understood as interchangeable.

The term “neoplasm” should be understood as a reference to a lesion,tumour or other encapsulated or unencapsulated mass or other form ofgrowth or cellular aggregate which comprises neoplastic cells. Theneoplastic cells comprising the neoplasm may be any cell type, derivedfrom any tissue, such as an epithelial or non-epithelial cell. Examplesof neoplasms and neoplastic cells encompassed by the present inventioninclude, but are not limited to central nervous system tumours,retinoblastoma, neuroblastoma, paediatric tumours, head and neck cancers(e.g. squamous cell cancers), breast and prostate cancers, lung cancer(both small and non-small cell lung cancer), kidney cancers (e.g. renalcell adenocarcinoma), oesophagogastric cancers, hepatocellularcarcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and isletcell tumours), colorectal cancer, cervical and anal cancers, uterine andother reproductive tract cancers, urinary tract cancers (e.g. of ureterand bladder), germ cell tumours (e.g. testicular germ cell tumours orovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelialcancers), carcinomas of unknown primary, human immunodeficiencyassociated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias,malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroidgland), mesothelioma and other pleural or peritoneal tumours,neuroendocrine tumours and carcinoid tumours.

In one particular embodiment, said neoplastic condition is a leukaemiaor lymphoma.

In another embodiment, said neoplastic condition is metastatic.

The subject undergoing treatment or prophylaxis may be any human oranimal in need of therapeutic or prophylactic treatment. In this regard,reference herein to “treatment” and “prophylaxis” is to be considered inits broadest context. The term “treatment” does not necessarily implythat a mammal is treated until total recovery. Similarly, “prophylaxis”does not necessarily mean that the subject will not eventually contracta disease condition. Accordingly, treatment and prophylaxis includeamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylaxis” may be considered as reducing the severity of theonset of a particular condition. “Treatment” may also reduce theseverity of an existing condition.

The present invention should therefore be understood to encompassreducing or otherwise ameliorating a condition in a mammal. This shouldbe understood as a reference to the reduction or amelioration of any oneor more symptoms of disease. Although it is always most desirable toachieve the cure of a disease, there is nevertheless significantclinical value in slowing the progression of a disease. For example, inthe context of a viral infection such as HIV or STD, even if completecure cannot be achieved, a reduction in the extent of viral load andspread may provide a means of controlling the infection such that thesevere immunodeficiency of HIV, for example, which is ultimately fatalis not experienced and a relatively normal life span can be achievedwithout the severe side effects that are characteristic of the currentanti-viral drug cocktails which patients are required to take. In thespecific context of neoplastic conditions, the T cells of the presentinvention, when administered to a patient, down-regulate the growth of aneoplasm. Reference to “growth” of a cell or neoplasm should beunderstood as a reference to the proliferation, differentiation and/ormaintenance of viability of the subject cell, while “down-regulating thegrowth” of a cell or neoplasm is a reference to the process of cellularsenescence or to reducing, preventing or inhibiting the proliferation,differentiation and/or maintenance of viability of the subject cell. Ina preferred embodiment the subject growth is proliferation and thesubject down-regulation is CD8⁺ T cell mediated killing. In this regard,the killing may be evidenced either by a reduction in the size of thetumour mass or by the inhibition of further growth of the tumour or by aslowing in the growth of the tumour. In this regard, and withoutlimiting the present invention to any one theory or mode of action, theneoplastic cells may be killed by any suitable mechanism such as directlysis or apoptosis induction or some other mechanism which can befacilitated by CD4⁺ or CD8⁺ T cells, or T cells lacking these CD4 andCD8 markers. The present invention should therefore be understood toencompass reducing or otherwise ameliorating a neoplastic condition in amammal. This should be understood as a reference to the prevention,reduction or amelioration of any one or more symptoms of a neoplasticcondition. Symptoms can include, but are not limited to, pain at thesite of tumour growth or impaired metabolic or physiological bodilyfunctions due to the neoplastic condition. It should be understood thatthe method of the present invention may either reduce the severity ofany one or more symptoms or eliminate the existence of any one or moresymptoms. The method of the present invention also extends to preventingthe onset of any one or more symptoms.

Accordingly, the method of the present invention is useful both in termsof therapy and palliation. To this end, reference to “treatment” shouldbe understood to encompass both therapy and palliative care. As would beunderstood by the person of skill in the art, although it is always themost desirable outcome that a neoplastic condition is cured, there isnevertheless significant benefit in being able to slow down or halt theprogression of the neoplasm, even if it is not fully cured. Withoutlimiting the present invention in any way, there are some neoplasticconditions which, provided they are sufficiently down-regulated in termsof cell division, will not be fatal to a patient and with which thepatient can still have a reasonable quality of life. Still further, itshould be understood that the present method provides a usefulalternative to existing treatment regimes. For example, in somesituations the therapeutic outcome of the present method may beequivalent to chemotherapy or radiation but the benefit to the patientis a treatment regime which induces either fewer side effects or ashortened period of side effects and will therefore be tolerated by thepatient much better. As detailed above, it should also be understoodthat the term “treatment” does not necessarily imply that a subject istreated until total recovery. Accordingly, as detailed above, treatmentincludes reducing the severity of an existing condition or ameliorationof the symptoms of a particular condition or palliation. In this regard,where the treatment of the present invention is applied at the time thata primary tumour is being treated it may effectively function as aprophylactic to prevent the onset of metastatic cancer. For example, forcertain types of solid tumours, it may still be most desirable tosurgically excise the tumour. However, there is always a risk that theentirety of the tumour may not be successfully removed or that there maybe escape of some neoplastic cells. In this case, by applying the methodof the present invention to lyse any such neoplastic cells, the methodis effectively being applied as a prophylactic to prevent metastaticspread.

In accordance with this aspect of the invention, the subject cells arepreferably autologous cells which are isolated and genetically modifiedex vivo and transplanted back into the individual from which they wereoriginally harvested. However, it should be understood that the presentinvention nevertheless extends to the use of cells derived from anyother suitable source where the subject cells exhibit a similarhistocompatability profile as the individual who is the subject oftreatment, so that the transferred cells can perform their function ofremoving unwanted cells, before being subjected to immune rejection bythe host. Accordingly, such cells are effectively autologous in thatthey would not result in the histocompatability problems which arenormally associated with the transplanting of cells exhibiting a foreignMHC profile. Such cells should be understood as falling within thedefinition of being histocompatible. For example, under certaincircumstances it may be desirable, necessary or of practicalsignificance that the subject cells are isolated from a geneticallyidentical twin, or from an embryo generated using gametes derived fromthe subject individual or cloned from the subject individual (in thiscase the cells are likely to correspond to stem cells which haveundergone directed differentiation to an appropriate somatic cell type).The cells may also have been engineered to exhibit the desired majorhistocompatability profile. The use of such cells overcomes thedifficulties which are inherently encountered in the context of tissueand organ transplants.

However, where it is not possible or feasible to isolate or generateautologous or histocompatible cells, it may be necessary to utiliseallogeneic cells. “Allogeneic” cells are those which are isolated fromthe same species as the subject being treated but which exhibit adifferent MHC profile. Although the use of such cells in the context oftherapeutics could result in graft vs host problems, or graft rejectionby the host, this problem can nevertheless be minimised by use of cellswhich exhibit an MHC profile exhibiting similarity to that of thesubject being treated, such as a cell population which has beenisolated/generated from a relative such as a sibling, parent or child orwhich has otherwise been generated in accordance with the methodsexemplified herein.

It would be appreciated that in a preferred embodiment the cells whichare used are autologous. However, due to the circumstances of a givensituation, it may not always be possible to generate an autologous stemcell population. This may be due to issues such as the urgency ofcommencing treatment or the availability of facilities to effecttransformation and directed differentiation. In this case, and asdetailed hereinbefore, it may be desirable or necessary to use syngeneicor allogeneic cells, such as cells which have been previouslytransfected and are available as frozen stock in a cell bank. Suchcells, although allogeneic, may have been selected for transformationbased on the expression of an MHC haplotype which exhibits lessimmunogenicity than some haplotypes which are known to be highlyimmunogenic or which has otherwise been generated in accordance with themethods exemplified herein.

Reference to an “effective number” means that number of cells necessaryto at least partly attain the desired effect, or to delay the onset of,inhibit the progression of, or halt altogether the onset or progressionof the particular condition being treated. Such amounts will depend, ofcourse, on the particular condition being treated, the severity of thecondition and individual patient parameters including age, physicalconditions, size, weight, physiological status, concurrent treatment,medical history and parameters related to the disorder in issue. Oneskilled in the art would be able to determine the number of cells of thepresent invention that would constitute an effective dose, and theoptimal mode of administration thereof without undue experimentation,this latter issue being further discussed hereinafter. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is preferred generallythat a maximal cell number be used, that is, the highest safe numberaccording to sound medical judgement. It will be understood by those ofordinary skill in the art, however, that a lower cell number may beadministered for medical reasons, psychological reasons or for any otherreasons.

As hereinbefore discussed, it should also be understood that althoughthe method of the present invention is predicated on the introduction ofgenetically modified cells to an individual suffering a condition asherein defined, it may not necessarily be the case that every cell ofthe population introduced to the individual will have acquired or willmaintain the subject modification and differentiation. For example,where a transfected and expanded cell population is administered intotal (i.e. the successfully modified or differentiated cells are notenriched for), there may exist a proportion of cells which have notacquired or retained the genetic modification and/or the desired T celldifferentiation. The present invention is therefore achieved providedthat the relevant portion of the cells thereby introduced constitute the“effective number” as defined above. However, in a particularlypreferred embodiment the population of cells which have undergonedifferentiation will be subjected to the identification of successfullymodified and differentiated cells, their selective isolation.

In the context of this aspect of the present invention, the subjectcells require introduction into the subject individual. To this end, thecells may be introduced by any suitable method. For example, cellsuspensions may be introduced by direct injection or inside a blood clotwhereby the cells are immobilised in the clot thereby facilitatingtransplantation. The cells may also be introduced by surgicalimplantation. This may be necessary, for example, where the cells existin the form of a tissue graft. The site of transplant may be anysuitable site, for example, subcutaneously. Without limiting the presentinvention to any one theory or mode of action, where cells areadministered as an encapsulated cell suspension, the cells will coalesceinto a mass. It should also be understood that the cells may continue todivide following transplantation. In this regard, the introduction of asuicide gene, as hereinbefore described, provides a convenient means ofcontrolling ongoing division.

The cells which are administered to the patient can be administered assingle or multiple doses by any suitable route. Preferably, and wherepossible, a single administration is utilised. Administration viainjection can be directed to various regions of a tissue or organ,depending on the type of treatment required.

In accordance with the method of the present invention, otherproteinaceous or non-proteinaceous molecules may be co-administered withthe introduction of the transfected cells. By “co-administered” is meantsimultaneous administration in the same formulation or in differentformulations via the same or different routes or sequentialadministration via the same or different routes. By “sequential”administration is meant a time difference of from seconds, minutes,hours or days between the transplantation of these cells and theadministration of the proteinaceous or non-proteinaceous molecules. Forexample, depending on the nature of the condition being treated, it maybe necessary to maintain the patient on a course of medication toalleviate the symptoms of the condition until such time as thetransplanted cells become integrated and fully functional (for example,the administration of anti-viral drugs in the case of an HIV patient).Alternatively, at the time that the condition is treated, it may benecessary to commence the long term use of medication to preventre-occurrence of the condition. For example, where the subject damagewas caused by an autoimmune condition, the ongoing use of a low level ofimmunosuppressive drugs may be required once the autoreactive cells havebeen destroyed.

It should also be understood that the method of the present inventioncan either be performed in isolation to treat the condition in issue orit can be performed together with one or more additional techniquesdesigned to facilitate or augment the subject treatment. Theseadditional techniques may take the form of the co-administration ofother proteinaceous or non-proteinaceous molecules or surgery, asdetailed hereinbefore.

Yet another aspect of the present invention is directed to the use ofstem cells or T cells differentiated therefrom, as hereinbefore definedin the manufacture of a medicament for the treatment of a conditioncharacterised by the presence of an unwanted population of cells in amammal.

In another embodiment, said stem cell is an iPSC or an HSC.

In still another embodiment, said stem cell is capable ofdifferentiating to a CD4⁺ T cell or a CD8⁺ T cell.

In still another embodiment, said TCR is an αβ TCR.

In yet still another embodiment, said stem cell such as iPSC is derivedfrom a T cell or thymocyte, preferably a CD8⁺ T cell or thymocyte.

In still another embodiment, the cell further comprises a nucleic acidmolecule encoding a non-signalling antigen-binding receptor, whereinsaid receptor comprises an antigen recognition moiety directed to CD47.

References made herein to “a cell” should be understood as referring toan isolated cell, or an isolated or substantially purified population ofcells. In reference to a cell population, by “substantially pure” it ismeant that a relevant cell type accounts for at least 70%, 75%, 80%,85%, 90%, 95%, 98%, 99% or greater percentage of all the cells in thecell population. For example, a cell population is substantially purefor a relevant T cell if such T cell accounts for at least 70%, 75%,80%, 85%, 90%, 95%, 98%, 99% or greater percentage of all the cells inthe cell population.

The present invention is further described by reference to the followingnon-limiting examples.

EXAMPLES

The present description is further illustrated by the following Exampleswhich demonstrate the development of certain embodiments of the presentinvention, including dual anti-cancer specific T cells, derived fromiPSC cells or HSCs. These examples should not be construed as limitingin any way.

Example 1: Enrichment of Cancer Peptide Antigen-Specific T Cells fromBlood WT-1 Specific TCR T-Cell Stimulation and Expansion

WT-1 specific T cells are very rare in normal human blood, but can beexpanded and enriched in order to be detected. In this context,peripheral blood mononuclear cells (PBMCs) were isolated usingFicoll-Hypaque density gradient centrifugation. Freshly isolated PBMCswere resuspended in tissue culture medium supplemented with human ABserum, L-glutamine and CD28 monoclonal antibody added to act as aco-stimulant of the T cells when WT-1 is present; anti-CD28 alonedoesn't activate T cells. PBMCs were then stimulated with Wilm's Tumor 1(WT-1) peptides overnight at 0.6 nmol/ml for each of the four WT-1peptides: WT-137 (VLDFAPPGA, SEQ ID NO: 22), WT-1126 (RMFPNAPYL, SEQ IDNO: 23), WT-1187 (SLGEQQYSV, SEQ ID NO: 24), and WT-1235 (CMTWNQMNL, SEQID NO: 25), which represent the main HLA Class I binding motifs. Datapresented in the examples of this application used WT-1 peptide 1-37 asrepresentative of this family of WT-1 peptides. WT-1 specific T cellscan be identified with HLA-WT-1 specific tetramers or by the earlyinduction of the surface molecule CD137 on stimulated but not resting Tcells. CD137 is a member of the tumor necrosis factor (TNF) receptorfamily. It is also known as 4-1BB. After 24-36 hours, CD137 positivecells (that is WT-1 stimulated T cells) were magnetically separatedusing a magnetic cell separator. CD137 positive (WT-1 specific TCR)cells were cultured in T cell expansion media consisting of X-Vivo-15base medium supplemented with human AB-serum, recombinant interleukin 7,interleukin 15 and interleukin 21. The corresponding CD137 negativecells were further subjected to CD3 magnetic separation. CD3 negativecells (predominantly B cells) were subjected to mitomycin C treatmentand used as WT-1 peptide-loaded antigen presenting feeder cells to theinduced CD137 positive population, while the remaining CD3 positivecells (non-WT-1 specific) were grown in culture to act as a control Tcell type for the down-stream functional assays. Media with therecombinant cytokines were replenished every second day.

For flow cytometry analysis, cells were resuspended in FACs Buffer: 30μl per 10⁶ cells. 10 μl of FcR blocking reagent was added to cells for 5mins at room temperature. 10 μl of HLA-A02 WT-1 tetramer was added andcells incubated for 20 mins at 4° C. protected from light. 50 μl of“T-Cell Activation” Cocktail was added and cells incubated for 20 minsat 4° C. protected from light. 100 μl of FACs Buffer was added plus 41of Aqua Amine and cells incubated for 5 mins, and subsequentlycentrifuged at 150×g for 5 mins. The supernatant was aspirated ordecanted and the pellet resuspended in 100 μl of BD Cytofix/Cytopermsolution per sample and cells incubated for 20 mins at 4° C. Cells werewashed in BD/Perm wash. IFN-γ antibody was diluted 1/100 in BD/Perm washsolution and incubated with cells for 30 minutes in the dark at 4° C.Cells were washed in BD/Perm wash and resuspended in FACs buffer priorto flow cytometric analysis. FACS data acquisition was done on aMiltenyi Quant cytometer.

T cells with a TCR specific for WT-1 peptide are normally very low infrequency (e.g., Schmeid et al (2015)) showed they are as few 1 per 10⁻⁶of CD8+ cells (range 3×10⁻⁷ to 3×10⁻⁶ cells). Following the stimulationprotocol described above, WT-1 TCR specific T cells increased ˜100 foldto ˜3.0% (WT-1 patient #1 1.5%; WT-1 patient #2 4.0%; FIG. 1).

Functional Analysis of WT-1 TCR T-Cells

The in vitro expanded T cells were additionally stimulated withautologous antigen presenting cells (B cells transformed with EBV) andprimed with the range of WT-1 peptides: WT-137 (VLDFAPPGA), WT-1126(RMFPNAPYL), WT-1187 (SLGEQQYSV), and WT-1235 (CMTWNQMNL). The T cellswere examined by flow cytometry for interferon gamma (IFNγ) productionusing the fluorescent bead assay. Cells were double labelled for WT-1peptide specificity via binding to a WT-1 peptide-HLA tetramer (see FIG.2).

The WT-1 stimulated T cells clearly expressed (80-90%) interferon gamma(IFNγ) (FIG. 2), a well recognised measure of T cell function (e.g.,Ghanekar et al (2001)). To potentially increase the level of CD8 T cellactivation (targeting WT-1 T cells), use was made of the LAG 3 inhibitorIMP 321. LAG3 is normally a “check point blockade”, inhibiting thestimulatory function of dendritic cells (DC) and the response to DC's asantigen presenting cells, by CD8 T cells. When added to the WT-1specific T cell activation assay, there was no effect of IMP 321 at 24hours, but after 4 days there was a doubling of the rare CD8+WT-1specific TCR T cells (FIG. 2H).

Example 2: Generation of iPSC from Human Blood T-Cells

For derivation of iPSC from human blood T cells, there are a number ofapproaches with varying levels of faithful retention of the original Tcell properties. iPSC have been produced from a broad repertoire ofperipheral blood T lymphocyte pool (T-iPSC) from a normal healthy human.The T-cells were pre-activated, for example, with the mitogen PHA oranti CD3 and anti CD28 antibodies. Using dual retroviral vectorcassettes each containing two of the Yamanaka reprogramming factors(Oct4, Sox 2, KLF, cMyc), multiple T-iPSC clones were generated whichwere validated at the cellular and molecular level, including flowcytometry and qRT-PCR for a range of markers including Nanog, Oct3/4,SSEA 3,4, TRA-1-60 and TRA-1-81. Their pluripotency was confirmed byteratoma formation after injection into NOD-SCID-IL common gamma chain−/− (NSGMice). Confirmation of T cell origin was confirmed by showingthat the TCR genes were rearranged.

The production of iPSC from WT-1 specific blood T cells is summarised inFIG. 3.

Example 3: Induction of Human T Cells from iPSC

This Example shows generation of genuine T cells from iPSC. These Tcells were shown to express the key features of typical T cells asnormally produced by the thymus. They were shown to express themainstream T cell αβTCR and CD8 with both β and α chains.

T cells have been induced from iPSC derived from whole adult blood Tcells or pre-selected CD8+ T cells, or antigen specific T cells (e.g.,those specific for WT-1) (T-iPSC), or adult fibroblasts. There are twobasic stages specialisation to haemopoiesis (haemopoietic stem cells or“HSC”) and partially lymphoid lineage, by culture on OP9 cells; transferof these cultured cells to OP cell line genetically modified to expressNotch signalling molecule Delta-like Ligand 1 (OP9-DL-L1) for thesubsequent induction of T cell differentiation.

Phase 1—Preparation of OP9 Support Cells and iPSC Colonies

Day −8: Mitomycin treated Mouse embryonic fibroblast feeder layers wereplated onto 0.1% gelatin coated TC plates at 0.3×10⁶ (14,250 cells/cm²)in 3 mL of MEF media (DMEM+15% FCS+1% pen/strep L-glutamine), andincubated overnight. OP9 cells were pre-prepared by plating onto 0.1%gelatin coated 10 cm TC plates at 0.25×10⁶ cells in 11 mL OP9 media(αMEM+20% FCS+1% pen/strep).

Day −7: iPS cells were thawed and plated onto the MEF cells, andincubated at 37° C. 5% CO₂ for 7 days.

Phase 2—Conversion of iPSCs to Haemopoietic Cells

Day 0: Start of haemopoietic specialisation. The iPS colonies weredissociated and plated onto OP9 for HSC differentiation. The colonysuspension was added dropwise for even distribution onto the OP9 plate.Fresh differentiation medium was added on days 1, 5 and 9.

Day 13 Harvest Induced HSC Precursors for T-Cell Differentiation

Cells cultured on the OP9 cell line were gently removed by Collagenase(working solution 100 μg/mL collagenase/HBSS; 37° C. for 1:15 hrs) andthe colonies further disrupted into single cells by trypsin/EDTA 0.05%at 37° C. for 30 mins. The cells were gently washed and examined byphase Contrast microscopy (FIG. 4) and by flow cytometry (FIG. 5). Thehaemopoietic nature of the cells was confirmed by flow cytometry (FIG.5).

Phase 3—Induction of iPSC-Derived HSC to T Cells

Day 13: Induction of T Cell Differentiation: Transfer of Day 13 OP9Conditioned (Haemopoiesis Induced) Cells to OP9DL-L1 Cells.

In a preferred embodiment, to enhance the efficiency of contact with theOP9DL-L1 cells, the OP9 conditioned cells were purified forCD34⁺CD43+(HSC) and then plated onto the OP9 DLL-1 cells for the firststage of T cell differentiation. A critical component of the processdisclosed herein was to collect the cells which initially grewunderneath the OP9 DL-L1 cells.

Cells collected from the OP9 cultures were resuspended in T celldifferentiation culture medium (OP9 media, SCF 5 ng/mL, Flt3 5 ng/mL,IL-7 5 ng/mL & Vitamin C 100 μM) and the suspension was added dropwiseto the OP9 DLL-1 cells and incubated at 37° C. Cells were harvestedafter 2, 9, 16, 23 and 30 days culture on the OP9 DL-L1 and subjected toflow cytometry analysis (FIG. 6 and FIG. 7).

When these cultures were examined for T cell development there was clearevidence of expression of the early markers CD7 and CD9 and the nextmarkers of T cell development with CD4 and CD8 expression (FIG. 7). Evenat this early stage there was already ˜10% of the cells expressing bothCD4+ and CD8+; these CD4+CD8+ cells are characteristic of T cells whichdevelop normally in the thymus cortex (Heng et al (2010)).

Flow cytometry showed progressive development of T cells from theinitial expression of CD5, CD7+ then CD8+. Most importantly the inducedT cells expressed the phenotype of “optimal, thymus produced” CD8 Tcells. They expressed the CD8β chain in addition to the CD8α chain(other reported T cell induction systems do not induce the optimal,signalling CD8β chain; e.g., Themeli et al (2013)). As indications offunction they also expressed CD3 with the αβTCR. Furthermore, thesecells were present as early as Day 16 of culture on OP9 DL-L1 cellscompared to day 30 in other reported systems.

Phase 4 Development of Mature T Cells

After a further 7 days (a total of 13 days on OP9 cells followed by 16days on OP DL-L1 cells), these developing T cells made a criticaltransition to expression of the T cell receptor complex with CD8+ Tcells clearly positive for CD3 and the αβTCR; in addition, these cellsexpressed the important CD8β—these are the desired cells for CAR-T.There was a corresponding further reduction in CD34+CD43+ HSC (FIG. 8).

This induction system has thus successfully produced mature CD8 T cellsfrom iPSC after 13 days culture on OP9 cells followed by 16 days onOP9DL-L1 cells.

Using the process described above, T cells expressing a TCR specific forWT-1 were produced from iPSC which were themselves derived from WT-1 TCRCD8+T cells (FIG. 9). These iPSC derived WT-1 T cells had a cytotoxicfunction equivalent to the original T cells from which the iPSC werederived (FIG. 10).

Example 4: Development of CAR Constructs

A component of Chimeric Antigen Receptor (CAR)-T cells is the antigenrecognition component of the CAR mediated by the scFv ectodomain,represented by a single-chain Fv (scFv) anchored by a CD8 or CD28 hingeand including a transmembrane (TM) region and the signal transduction ofthe CAR via the cytoplasmic endodomain—represented by CD28, 4-1BB andthe CD3 zeta (CD3ζ) chain. There are also two suitable viral deliverysystems—retrovirus and lentivirus. Exemplary CAR and CD47-bindingreceptor constructs are shown in FIG. 11.

Example 5: Chimeric Antigen Receptor Vector Cloning Strategies

Exemplary chimeric antigen receptor vector cloning strategies areillustrated in FIGS. 12-13. FIG. 14 shows our 2^(nd) generation CAR andthe strategy for a non-signalling anti-CD47 construct. Exemplarysequences of chimeric antigen receptors, non-signalling antigen-bindingreceptors, and the various domains thereof, are provided in SEQ ID NOS:1-20.

Example 6: Chimeric Antigen Receptor Transduction of T Cells LentivirusProduction

293T cells were plated onto poly-L-lysine (Sigma) coated 175 cm² flasks.Two hours prior to transfection, medium was replaced with DMEMsupplemented with 10% FCS. The lentiviral transfer vector DNA, togetherwith packaging and envelope plasmid DNA were combined and mixed withLipofectamine2000. The solution was briefly vortexed and incubated atroom temperature for 30 min. Following this, the solution was mixedagain and then added dropwise to the cells. Flasks were returned to theincubator. Six hours later, fresh growth medium added. Viral supernatantwas collected after 48 hrs and cleared by centrifugation at 1500 rpm for5 min at 4° C. then passed through a 0.45 μm pore PVDF Millex-HV filter(Millipore). Concentration of lentivirus using ultracentrifugation wasperformed with a Sorval Discovery 100 SE centrifuge using an AH-629rotor. 30 mL of filtered virus supernatant was added to 36 mLpolyallomer conical tubes (Beckman). Centrifugation was performed for 90min at 20,000 g. Supernatant was completely removed and virus pelletsresuspended in 300 μL PBS and stored at −80° C. until use.

Generation of CAR-T Cells

FIG. 11 and SEQ ID NOS: 1-6 show a panel of Chimeric Antigen Receptor(CAR) and CD47-binding receptor constructs that have been developed—withscFv specific for either TAG 72 or CD19 (as a positive control). Theseconstructs use either human CD8 or CD28 as hinge region and CD28, CD3ζchain or 4-1BB cytoplasmic activation signalling domains. CAR andCD47-binding receptor constructs are cloned into lentiviral vectors asdescribed in the previous paragraph.

Optimal lentiviral transduction of T cells involves their activation atthe TCR and co-stimulatory receptors. Accordingly, on day 0, fresh PBMCwere collected by apheresis from healthy donors, were enriched foractivated T cells with the use of anti-CD3 and anti-CD28 antibodiesbound to paramagnetic beads (Dynabeads ClinExVivo CD3/CD28, Invitrogen,Camarillo, Calif., USA) at a ratio of 3:1 (beads:cells). The cells andbeads were co-incubated for 1 h at room temperature, andCD3+ cellenrichment was performed with the use of magnet (Invitrogen). Cells inthe CD3+ fraction were resuspended in initiation media at aconcentration of 1×10⁶ cells/ml in T cell expansion medium with 100IU/ml IL-2. On day 1, RetroNectin was used to coat cell culture dishesat a concentration of 2 mg/cm2 in a solution of 10 mg/mL in PBSovernight at 4° C. On day 2, the RetroNectin solution was aspirated andthe same volume of blocking solution, consisting of 0.5% human serumalbumin in PBS, was added to each bag and incubated at room temperaturefor 30 min. The blocking solution was aspirated, and each bag washedwith PBS. Lentiviral supernatant was rapidly thawed and added to eachdish with T cell expansion medium with 300 IU/ml IL-2. The cultures wereplaced back into the incubator and left for at least 24 h. On day 4, thetransduction was stopped; cells were resuspended in fresh T-cellexpansion medium at a concentration of 0.5-1×10⁶ cells/mL. The cultureswere maintained until day 14 and fed every other day with freshexpansion media to maintain cell concentration at 1×10⁶ cells/mL.

Initially blood derived human T cells were subjected to CARtransduction, the success of which was measured by flow cytometry,depicting the eGFP+ cells (FIG. 15). This was also confirmed by WesternBlot analysis (FIG. 16).

Assessing the Functionality of the CAR-T Cells

The TAG-72 CAR-T cells (created from normal PBMC) were examined fortheir ability to kill TAG72 expressing target cancer cells in vitro. Thereal time cell monitoring system (xCELLigence) was employed to determinethe killing efficiency of CAR-T cells in vitro. 10,000-2×10⁶ targetcells/1004, (for example the TAG72+ ovarian cancer cell line CaOV4) weredeposited into RTCA plates. In some instances, tethering of target cellsby anti-hCD40 or by human fibronectin pre-coating of the plate may berequired. Target cells are maintained at 37° C., 5% CO₂ for 3-12 h toallow for cellular attachment. Following attachment of target cells,CAR-T effector cells were added at variable effector:target ratios(ranging from 1:1 to 10:1). In some experiments, CAR-T effector cellswere isolated based of GFP expression of CAR-T cells via FACS prior touse. Co-cultures were maintained in optimal growth conditions for atleast 12 h. Cellular impedance was monitored throughout; a decrease inimpedance is indicative of cell detachment and ultimately cell death.

FIG. 17 shows results from this experiment monitored over 40 hours. Theovarian cancer cell line CaOV4 grew consistently over this time period(blue line). In contrast, cultures supplemented with TAG-72 specific CART cells showed an initial growth phase that was significantly less thanthat of target cells alone, followed by gradual elimination of thetarget cells over time (purple line). To overcome the non-specifickilling due to CD3/CD28 activation, the TAG 72 CAR-T cells were isolatedby flow cytometry and compared to CD19 CAR-T cells and non-CAR-Tcells—without prior CD3/CD28 activation (FIG. 21). The data shown inFIG. 21 indicate strong antigen specificity of TAG-72 CAR-T cells in thefirst 24 hours of culture with TAG-72 expressing cancer cells, sincenegative controls of vector only transfected T cells and non-transfectedT cells showed no killing of the cancer cells in this time frame.

The above studies were performed on polyclonal T cells derived fromperipheral blood. To demonstrate CAR-transduction of mono-specific Tcells expressing a TCR specific for a nominal cancer peptide antigen,WT-1 TCR specific T cells derived from iPSC formed from WT-1 specificTCR, were transduced by TAG 72 CAR lentivirus. FIG. 22A shows successfulCAR transduction of these WT-1 specific TCR CD8+ T cells themselvesderived from iPSC produced from WT-1 specific T cells. The CAR containedthe specificity for TAG 72. Most importantly, FIG. 22B shows thesuccessful transduction of WT-1 specific TCR CD8+ T cells themselvesderived from iPSC produced from WT-1 specific T cells, with a CARconstruct for both TAG 72 and CD47. This indicates that T cells can beproduced with three specificities for cancer: WT-1 (TCR), TAG 72 (CAR)and CD47 (truncated, CD47-binding receptor).

The results demonstrate the development of dual specific CAR-transducedcancer specific TCR (WT-1) derived from iPSCs, which were themselvesderived from WT-1 specific TCR T cells from normal adult blood.

FIG. 20 shows that both components of the dual specific T cells(containing the WT-1 TCR and the TAG72 CAR) can contribute to thekilling of cancer cells. When corrected for spontaneous cell death, evenat low effector-target ratio (here it is 2 effectors to 1 target cell)the WT-1 cells caused approximately 10% cell killing and then additionof the TAG72 CAR by transduction caused an additional 10% killing.

Example 7: Chimeric Antigen Receptor Transduction of iPSC

The production of multi-specific CAR-T cells can be achieved by multipleapproaches including CAR transduction of existing blood T cells (FIG.15) or by transduction of iPSC which are then induced to T cells(expressing cancer specific TCR and the CAR's) (e.g., SEQ ID NOS: 1-6).Multiple iPSC lines have been used to progress with CAR-T transduction.These iPSC could be derived from non-T cells, or from cancer antigenspecific T cells (e.g., WT-1) which would retain the TCR generearrangements. These iPSC were either derived from adult fibroblasts orfrom T cells with an endogenous TCR specific for a specific cancerantigen (WT-1 peptide).

The iPSC were stably transduced with a single cistron, as shown in FIG.14 in which the CAR ectodomain comprises an scFv specific for TAG72 (oras control CD19). The hinge (Stalk) region and the transmembrane regionare derived from CD28 or CD8 and the cytoplasmic endodomain, whichcomprises T cell signal transduction domains, is derived from CD28 andTCR chain. The CAR has a C-terminal extension encoding EGFP linked by aP2A self-cleaving polypeptide to separate the CAR and reporter.Following viral integration, the P2A was cleaved and the success oftransduction was quantified by measuring the fluorescent of the releasedEGFP reporter. GFP fluorescence illuminates the success of transduction.It can be used to show transduction in situ (FIG. 21) or to identify andisolate CAR transduced iPSC via flow cytometry (FIG. 22, 23).

These studies clearly show the ability to transduce iPSC withlenti-virus CAR constructs. FIG. 21A shows the successful transductionof iPSC derived from human fibroblasts with a CAR encoding TAG 72 orCD19 (FIG. 21A). FIG. 21B shows the successful transduction of iPSCderived from WT-1 TCR specific T cells, with TAG72. Any T cell derivedfrom this line will thus express dual anti-cancer specificity (WT-1 viaTCR; TAG 72 via CAR).

It is also possible to isolate the transduced iPSC by fluorescent-basedcell sorting. The positive cells can be collected and replated tosuccessfully form (CAR transduced) iPSC colonies (FIG. 24).

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

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1.-74. (canceled)
 75. A composition, comprising: (i) a nucleic acidencoding a first chimeric antigen receptor (CAR) which comprises anantigen recognition moiety operably linked to a T cell activation moietythrough a hinge region and a transmembrane domain and directed to afirst antigenic determinant; and (ii) a nucleic acid encoding anantigen-binding receptor lacking a functional signaling intracellulardomain which comprises an antigen recognition moiety, a hinge and atransmembrane region of a CAR, directed to a second antigenicdeterminant.
 76. The construct of claim 75, wherein the hinge region inthe antigen-binding receptor has cysteine residues either removed orsubstituted to prevent the formation of dimers.
 77. The constructaccording to claim 75, wherein the hinge region in the antigen-bindingreceptor is selected from a CD8 hinge and a CD28 hinge.
 78. Theconstruct according to claim 75, wherein the hinge region in the CARcomprises one or more cysteine residues to direct dimerization of theCAR.
 79. The construct according to claim 75, wherein the hinge regionin the CAR is selected from a CD8 hinge and a CD28 hinge.
 80. Theconstruct according to claim 75, wherein the first antigenic determinantis selected from TAG-72, CD19 and MAGE.
 81. The construct according toclaim 75, wherein the second antigenic determinant is CD47.
 82. Theconstruct of according to claim 75, wherein the first antigenicdeterminant is TAG-72 and the second antigenic determinant is CD47. 83.The construct according to claim 75, wherein the nucleic acid encodingthe CAR is operably linked to the nucleic acid encoding theantigen-binding receptor through a nucleotide sequence encoding aself-cleaving peptide.
 84. A cell or a population thereof, wherein (i)the cell is (a) stem cell or (b) a derivative cell obtained fromdifferentiating the cell of (a); and (ii) the cell comprises theconstruct as defined in claim
 75. 85. The cell according to claim 84,wherein the cell expresses at least one homozygous HLA haplotype. 86.The cell according to claim 84 for use in the treatment of a neoplasticcondition, a microorganism infection, or an autoimmune condition.